CN107312062A - Stimulating responsive polymer for purifying biological molecule - Google Patents

Stimulating responsive polymer for purifying biological molecule Download PDF

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Publication number
CN107312062A
CN107312062A CN201710282087.XA CN201710282087A CN107312062A CN 107312062 A CN107312062 A CN 107312062A CN 201710282087 A CN201710282087 A CN 201710282087A CN 107312062 A CN107312062 A CN 107312062A
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polymer
stimulating responsive
solution
responsive polymer
sample
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CN107312062B (en
Inventor
J·贾比尔
W·莫亚
J·哈姆日克
A·布迪夫
Y·张
N·索伊斯
J·查库迪恩
N·辛格
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EMD Millipore Corp
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Millipore Corp
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    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01JCHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J41/00Anion exchange; Use of material as anion exchangers; Treatment of material for improving the anion exchange properties
    • B01J41/08Use of material as anion exchangers; Treatment of material for improving the anion exchange properties
    • B01J41/12Macromolecular compounds
    • B01J41/14Macromolecular compounds obtained by reactions only involving unsaturated carbon-to-carbon bonds
    • CCHEMISTRY; METALLURGY
    • C08ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08FMACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
    • C08F26/00Homopolymers and copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and at least one being terminated by a single or double bond to nitrogen or by a heterocyclic ring containing nitrogen
    • C08F26/02Homopolymers and copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and at least one being terminated by a single or double bond to nitrogen or by a heterocyclic ring containing nitrogen by a single or double bond to nitrogen
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K1/00General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14Extraction; Separation; Purification
    • C07K1/30Extraction; Separation; Purification by precipitation
    • C07K1/32Extraction; Separation; Purification by precipitation as complexes
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01JCHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J41/00Anion exchange; Use of material as anion exchangers; Treatment of material for improving the anion exchange properties
    • B01J41/08Use of material as anion exchangers; Treatment of material for improving the anion exchange properties
    • B01J41/12Macromolecular compounds
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01JCHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J41/00Anion exchange; Use of material as anion exchangers; Treatment of material for improving the anion exchange properties
    • B01J41/08Use of material as anion exchangers; Treatment of material for improving the anion exchange properties
    • B01J41/12Macromolecular compounds
    • B01J41/13Macromolecular compounds obtained otherwise than by reactions only involving unsaturated carbon-to-carbon bonds
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K1/00General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14Extraction; Separation; Purification
    • C07K1/30Extraction; Separation; Purification by precipitation
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K16/00Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • CCHEMISTRY; METALLURGY
    • C08ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08FMACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
    • C08F126/00Homopolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and at least one being terminated by a single or double bond to nitrogen or by a heterocyclic ring containing nitrogen
    • C08F126/02Homopolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and at least one being terminated by a single or double bond to nitrogen or by a heterocyclic ring containing nitrogen by a single or double bond to nitrogen
    • CCHEMISTRY; METALLURGY
    • C08ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08FMACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
    • C08F26/00Homopolymers and copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and at least one being terminated by a single or double bond to nitrogen or by a heterocyclic ring containing nitrogen
    • C08F26/02Homopolymers and copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and at least one being terminated by a single or double bond to nitrogen or by a heterocyclic ring containing nitrogen by a single or double bond to nitrogen
    • C08F26/04Diallylamine
    • CCHEMISTRY; METALLURGY
    • C08ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08FMACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
    • C08F8/00Chemical modification by after-treatment
    • CCHEMISTRY; METALLURGY
    • C08ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08FMACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
    • C08F8/00Chemical modification by after-treatment
    • C08F8/02Alkylation
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53Immunoassay; Biospecific binding assay; Materials therefor
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K2317/00Immunoglobulins specific features
    • C07K2317/10Immunoglobulins specific features characterized by their source of isolation or production

Abstract

The present invention relates to the stimulating responsive polymer for purifying biological molecule, the stimulating responsive polymer is soluble stimulating responsive polymer, it is selected from the group that the polymer containing allyl amine and copolymer being modified by the polymer and copolymer containing allyl amine and benzyl are constituted, wherein the polymer can be combined when adding and stimulating and deposit sample in biomolecule of interest.Present invention also offers the method that the target molecule made using the polymer in sample is separated with one or more impurity, it comprises the following steps:(a) sample comprising target molecule and one or more impurity is provided;(b) sample is contacted with the soluble stimulating responsive polymer of claim 1, is consequently formed the complex compound of polymer and one or more impurity;In the sample add stimulate, thus from solution be settled out the complex compound, target molecule with one or more impurity is separated (c).

Description

Stimulating responsive polymer for purifying biological molecule
The application be Chinese Patent Application No. be on May 16th, 2,011 201180024500.3 applyings date Chinese patent The divisional application of application.
Priority data
This application claims the priority of U.S. Provisional Patent Application No.61/395769 filed in 17 days Mays in 2010 power Benefit, entire contents are incorporated herein by reference.
Invention field
The present invention relates to the polymer for protein purification.Particularly, the present invention is related to stimuli responsive at least in part Property polymer, it is used for the purification of target molecule from the sample containing target molecule and one or more impurity.
Background of invention
Effective and economic large scale purification biomolecule such as such as therapeutic protein (including antibody) is biological The aspect become more and more important on technology and medical industry.Generally, the purification process is quite fine and expensive, and including being permitted More different steps.For example typically in the case of protein, protein is produced using cell culture method, example Such as using mammal or bacterium cell line, it is by inserting the restructuring containing gene (it encodes described protein) Cytosome is engineered, and produces protein of interest.Generally, after target protein expression, by it and one kind or many Plant undesirable component (including such as host cell proteins matter, culture medium accessory substance and DNA) and be broken up into difficult challenge.When The therapeutic protein is intended for use in the mankind, and when having to pass through FDA (Food and Drug Adminstration) (FDA) approval, it is such Separation is especially important.
Generally, currently used for protein separation and/or purification process includes at least following step:Pass through lysis To reclaim intracellular protein or protein reclaimed from culture medium in the case of secretary protein;Using differential from Heart method or filtration method remove cell and cell fragment, to obtain containing protein purification sample of interest;With many Protein of interest in sample and different impurities are separated using a variety of chromatographic medias in one step process.
Different types of polymer (including polyelectrolyte) is used in one or more steps to carry out purifying biological point Son, particularly protein.It is known (see, for example, the world to carry out protein purification using polyelectrolyte for example in flocculating PCT Patent Application No.WO2008/091740).This can be completed with extensive polymer, and unique required general spy Property be the polymer must have and material (such as target molecule or impurity) a certain degree of interaction of interest.Most Common method is to use such as polyelectrolyte of the polymer containing ionic species.Generally, polyelectrolyte is added to protein In mixture, and purifying is realized via the selective flocculation of one or more components of the mixture.This scheme One crucial shortcoming is to must be added to the polyelectrolyte of careful controlled quentity controlled variable to avoid residual polymer pollution from (for example working as polymer Amount it is too high when) or avoid invalid flocculating (such as when the amount of polymer is too low).Because ion exchange and others Electrically charged chromatographic media is generally used in protein purification, therefore remaining polyelectrolyte can be potentially with reference to the pure of downstream Change medium used in step, thus pollute methods described and cause methods described to become complicated.
Recently, such technique has been developed, has been which overcomed and to carry out purifying biological molecule relevant one using polymer A little challenges (disclosing No.WO2008/079302A2 see, for example, International PCT).Stimulating responsive or " thorn are for example developed Quick property (Smart) " polymer, it can be attached to soluble (such as host cell proteins matter, DNA, cell culture addition Agent) and insoluble (such as cell and cell fragment) both components it is upper (see, for example, U.S. Publication No.20080255027 and 20090036651).It is such in face of widely using although stimulating responsive polymer shows generally bigger prospect One crucial challenge of polymer is to exist from laboratory scale to big production-scale different scales to implement Simple stimulation.
The content of the invention
The present invention provides the new stimulating responsive polymer based on polyelectrolyte, and its is easily scalable, and in wide pH Carried out with the range of conductivity, so that they can be used in purifying extensive biomolecule, including such as human cytokines Matter.
In some embodiments of the present invention there is provided a kind of stimulating responsive polymer, it includes polyelectrolyte master Chain, the main chain includes one or more hydrophobic groups, wherein after stimulation is added, the polymer can combine and precipitate sample Biomolecule of interest in product.
In some embodiments, the polyelectrolyte main chain of Inventive polymers includes at least two monomeric units or extremely Few three monomeric units.In some embodiments, at least 50% monomeric unit includes electric charge.In other embodiments In, each monomeric unit of the polyelectrolyte main chain includes electric charge.
In some embodiments, stimulating responsive polymer of the invention includes polyamine main chain.In some embodiments In, one or more hydrophobic groups are phenyl.
The stimulating responsive polymer of the present invention can be used to purifying desired target molecule, and by by desired target One or more undesirable materials that molecule is present in together with the desired target molecule in sample separate to carry out so Purifying.
Therefore, in some embodiments, stimulating responsive polymer of the invention combines and precipitated life of interest Thing molecule, itself is the desired target molecule for combining and precipitating by the stimulating responsive polymer.In other implementations In scheme, stimulating responsive polymer combines and has precipitated biomolecule of interest, and it is and desired target molecule one Act the undesirable material being present in sample.
In some embodiments, the biomolecule of interest is therapeutical peptide (that is, desired target molecule). In some embodiments, the therapeutical peptide is antibody (such as monoclonal antibody).
In other embodiments, the biomolecule of interest is selected from host cell proteins matter, DNA, RNA, lipid, disease Poison, endotoxin, cell culture additive, full cell and cell fragment.
In some embodiments, polymer of the invention is to stimulating being in response to property, and the stimulation forms complex compound Salt.
Present invention additionally comprises the method using polymer described herein.The stimulating responsive polymer is relative to existing skill Polymer described in art is unique and creative, shows that they replace or improved one in purification process Or multiple steps, thus significantly improve the desired target point from one or more undesirable material purifying or separation The overall purity of son.
Therefore, in some embodiments there is provided a kind of method for improving target molecule purity, wherein this method is included Step:(a) sample comprising target molecule and one or more impurity is provided;(b) by the sample with including polyelectrolyte main chain The stimulating responsive polymer of (it includes one or more hydrophobic groups being connected on main chain) is suitable for the polymer knot Close and contact under the conditions of first group of target molecule in solution, thus form the complex compound of polymer and target molecule;(c) The addition stimulation in the sample under the conditions of second group suitable for complex compound is precipitated out from solution, the wherein complex compound Precipitation result in the separation of target molecule and one or more impurity, which thereby enhance the purity of target molecule.
In some embodiments of the inventive method, this method is further comprising the recovery target molecule from complex compound Step.
In an alternate embodiment, stimulating responsive polymer of the invention combines and has precipitated one or more miscellaneous Matter, rather than target molecule, the precipitation of the wherein complex compound of the polymer and one or more impurity result in target molecule and one The separation of kind or plurality of impurities, which thereby enhances the purity of the target molecule.Therefore, such method includes step:(a) provide Sample comprising target molecule and one or more impurity;(b) by the sample with comprising polyelectrolyte main chain, (it is comprising being connected to One or more hydrophobic groups on main chain) stimulating responsive polymer a kind of or many suitable for the polymer is attached to Contacted under the conditions of first group on kind of impurity, thus form the complex compound of polymer and one or more impurity;Suitable (c) It will be stimulated under the conditions of second group of the complex compound is precipitated and add the sample, the precipitation of the wherein complex compound result in target molecule With the separation of one or more impurity, the purity of target molecule is which thereby enhanced.
In some embodiments, stimulating responsive polymer of the invention includes following structure:
Wherein x and y represent the monomeric unit of the polymer;R1And R2It is charged groups, which form polyelectrolyte master A part for chain (B);And R3It is attached to the hydrophobic group in the charged groups of main chain.Y monomeric units are (that is, with even The hydrophobic group being connected on main chain) represent the polymerization with the ratio of monomeric unit total (that is, x and y monomeric units summation) " the hydrophobic modified percentage " of thing.
In some embodiments, stimulating responsive polymer of the invention includes following structure:
Wherein x, y and z are the monomeric units of the polymer;R1、R2And R3It is charged groups, which form the poly- electrolysis A part for matter main chain (B);R4It is attached to the hydrophobic group in the charged groups of main chain;And R5It is attached to main chain Functional group in charged groups.Y monomeric units (that is, with the hydrophobic group being connected on main chain) and monomer in polymer The ratio of the number of the sum (that is, x, y and z monomeric unit summation) of unit represents " the hydrophobic modified percentage of the polymer Rate ".In addition, the sum of z monomeric units (that is, with the functional group being connected in the charged groups of main chain) and monomeric unit The ratio of the number of (that is, x, y and z unit summation) represents " the functional group modification percentage " of the polymer.
Generally, it should be understood that the polymer included by the present invention can have any monomer described here of " n " number Unit x, y or z, n is equal to or more than 2 here.
In other embodiments still, stimulating responsive polymer of the invention includes following structure:
Wherein x and y represent monomeric unit;R1、R2It is aliphatic amido (such as primary amine or secondary amine and/or aromatic amine), its Form the part containing carbon backbone chain of polyelectrolyte;And R3It is attached to amido R2On hydrophobic group, and include 4 Or more carbon atoms (such as alkyl, alkenyl, arylalkenyl or fluorocarbon group).In some embodiments, y (that is, has The monomeric unit for the hydrophobic group being connected in the charged groups of the polyelectrolyte main chain) and x (that is, polyelectrolyte masters The unmodified charged groups of chain) ratio be 0.01-0.75 or 0.05-0.75.Therefore, hydrophobic group is modified percentage Rate will be total polyelectrolyte monomeric unit (that is, x+y) 1%-75% or 5%-75%.
In other embodiments still, stimulating responsive polymer of the invention includes following structure:
Wherein R1、R2And R3It is aliphatic amine group, which form a part (such as primary amine of carbon containing polyelectrolyte main chain Or secondary amine and/or aromatic amine);And R4The hydrophobic group containing 4 or more carbon atoms, and selected from alkenyl, Aralkyl and arylalkenyl;And R5It is the hydrophobic group containing 4 or more than 4 carbon atoms, and selected from alkyl or fluorine carbon Group.The total ratio of y monomeric units and polyelectrolyte monomeric unit is 0.01-0.75.Z monomeric units and polyelectrolyte list The ratio of body unit sum is 0.05-0.5 or 0.01-0.5.Therefore, hydrophobic group be modified percentage be 1%-75% or Person 5%-75%, and functional group modification percentage are 1%-50% or 5%-50%.
Include such method using the other method of the stimulating responsive polymer of the present invention, it being capable of purification of target Molecule or product of interest (such as antibody), while making the amount of residual polymer in the sample minimum.
In some embodiments there is provided a kind of stimulating responsive polymer using the present invention, by target molecule (example Such as antibody) separated with one or more impurity, while so that the minimum method of the residual volume of polymer, wherein this method includes step Suddenly:(a) sample comprising target molecule and one or more impurity is provided;(b) sample and stimulating responsive polymer are existed Suitable for being contacted under the conditions of the polymer is attached on one or more impurity first group, thus polymer and one kind are formed Or the first complex compound of plurality of impurities;Wherein first group of condition is included in before or after adding the polymer, adjusts sample The pH or salinity of product;(c) the first complex compound is precipitated from sample under a second set of conditions;(d) by the sample and multivalence from Son contact, thus forms the second complex compound of residual polymer and multivalent ion;Precipitate second complex compound (e);(f) Target molecule is reclaimed from sample;Thus the target molecule in sample is separated with one or more impurity, while reducing remaining The amount of polymer in the sample.
In some embodiments, the target molecule is antibody.In a kind of specific embodiment, the antibody is Dan Ke Grand antibody.
In a kind of specific embodiment, being walked comprising chromatography for target molecule is reclaimed in the different method of the present invention Suddenly.In an alternate embodiment, reclaim target molecule and include filtration step.
In some embodiments, method of the invention can be including the use of the stimulating responsive polymer of the present invention two Individual or more steps.Stimulating responsive polymer can be for example used to precipitate a kind of in a step of purification process Or plurality of impurities, and same or different polymer can be used to precipitate target in the different step of methods described Molecule or desired product.
In some embodiments, one or more impurity are selected from host cell proteins matter, DNA, RNA, antibody and gathered Collective, virus, endotoxin, full cell, cell fragment and cell culture additive.
Brief description of the drawings
Fig. 1 is a schematic diagram, illustrates the reaction of polyallyl amine polymer and benzyl chloride.
Fig. 2 illustrates the reaction scheme for the PAH (HC-t-BuMPAA) that caproic acid and the tert-butyl group are modified.
Fig. 3 is a figure, what the effect stimulated which show sodium chloride for multivalent ion and caproic acid and the tert-butyl group were modified The pH responses of PAH (HC-t-BuMPAA).X- axles represent pH, and Y- axles represent centrifuge separating liquid (centrate) The turbidity of (that is, the output of centrifuge).
Fig. 4 illustrates the synthesis of the polyvinylamine described in embodiment 29.
Fig. 5 illustrates the deprotection reaction (deprotection) of polyamine after polymerization.
Fig. 6 illustrates the reaction of polyvinylamine and benzyl chloride.
Fig. 7 illustrates the polymerization and reaction scheme for forming multivalent ion stimulating responsive copolymer.
Fig. 8 illustrates the NMR spectra of the polyvinylamine (PVA) of the modification of embodiment 35.1H NMR integration shows benzyl Base modification degree is about 18%.
Fig. 9 illustrates the NMR spectra of the PAH of the modification of embodiment 36.1H NMR integration shows that benzyl is modified Degree is about 33%.
Figure 10 illustrates a figure, and which confirms non-stimulated responsive polymer (such as chitosan) and stimulating responsive are poly- Influence of the dose of polymer of compound (PAH that for example benzyl is modified) for centrifuge separating liquid turbidity.X- axles are poly- Compound dosage (wt%), and Y- axles are centrifuge separating liquid turbidity (NTU), as described in embodiment 37.
Figure 11 illustrates a figure, and which confirms exist and gather in the absence of for benzyl PAH stimulating responsive During the stimulation of compound, the influence of dose of polymer.X- axles are dose of polymer wt%, and Y- axles are centrifuge separating liquid turbidity (NTU), as described in embodiment 38.
Figure 12 illustrates a kind of typical scheme for purifying biological molecule.
Figure 13 illustrates a kind of purification schemes, and it includes being used to improve the stimulating responsive polymerization of the purifying of cell culture Thing.The stimulating responsive polymer eliminates one or more impurity, still, and the polymer does not combine desired target point Son.
Figure 14 illustrates a kind of purification schemes, and it includes being used to improve the stimulating responsive of the clarifying process of cell culture Polymer.The stimulating responsive polymer removes one or more impurity via flocculating, still, and the polymer does not combine institute Desired target molecule, and remove residual polymer by adding stimulation after clarification.
Figure 15 illustrates a kind of purification schemes, and it includes being used to improve the stimulating responsive of the clarifying process of cell culture Polymer.The stimulating responsive polymer removes one or more impurity, still, the polymer not combining target molecule, and By other sorbent filter step after clarification, to remove residual polymer.
Embodiment
The invention provides at least one of new and improved stimulating responsive polymer, it is included with one or many The polyelectrolyte main chain that individual hydrophobic group is modified, the wherein solubility of the polymer can be changed by adding stimulation.
Stimulating responsive polymer described herein and using its method than described in prior art these are more effective, its Show that they provide improved pH scopes, include such as protein for purifying desired target molecule, and remove not It is desired material such as impurity, such as host cell proteins matter, DNA, RNA, lipid, endotoxin, cell culture additive, thin Born of the same parents and cell fragment.In some embodiments, simple the being in response to property of multivalent salts of polymer described herein for low concentration , thus allow to improve property on a large scale and reduction conductivity relative to existing salt responsive polymer.In different implementation In scheme, polymer of the invention can effectively remove the full cell from cell culture medium, cell fragment and other can Solubility impurity.The polymer also can effectively remove the protein containing protein of interest and one or more impurity and mix Impurity in compound.In addition, different polymer described herein can effectively capture the target molecule in sample and be closed Protein/product of note, thus separates them with one or more impurity present in sample, and improve target molecule Purity.
Present invention additionally comprises extensive condition is used, carry out purification of target molecule using polymer described herein and for example treat Property method of protein.
Be not intended to be limited to theory, it is contemplated that stimulating responsive polymer described herein can be used in combine and Precipitate desired target molecule such as therapeutic protein or desired product or undesirable material is for example a kind of Or plurality of impurities, including such as host cell proteins matter, DNA, RNA, lipid, endotoxin, cell culture additive, full cell And cell fragment.Generally, the molecule combined by the polymer of the present invention is referred to as biomolecule of interest, is the phase but regardless of it The target molecule of prestige or undesirable material.
As described here, the selection of the specific stimulating responsive polymer used for plan is beaten based on the polymer Calculate and what is combined to determine.For example (have in the case of biomolecule of interest when it is in the pH higher than its pI net Negative electrical charge (such as full cell, cell fragment, DNA, endotoxin and protein)), it may be desirable to use and include polyelectrolyte main chain Stimulating responsive polymer, it is cation (that is, positively charged).On the other hand, biomolecule of interest (its There is net positive charge (such as protein) during pH less than its pI) in the case of, it may be desirable to using including polyelectrolyte master The stimulating responsive polymer of chain, it is anion (that is, negatively charged).
Under conditions of used in the process of the purification process positive charge be polymer it is intrinsic, or positive charge can use pH Change produce, described stimulating responsive polymer of changing into imparts electric charge.
A kind of important parameter for influenceing the recycled in its entirety rate of biomolecule is hydrophobic modified in the polyelectrolyte main chain Group and the ratio of remaining unmodified charged groups.For example, with the increase of hydrophobic group percentage, by non-specific Property the formed biomolecule of interaction loss also increase.So, for given biomolecule, band can be used The specific ratios of charge groups and hydrophobic group cause the biomolecule rate of recovery maximum.In addition, high hydrophobic group hundred Ratio is divided to limit the effect of the solubility and charged groups of polymer on the polyelectrolyte main chain.
In addition, the modification of the electrically charged amido benzyl chloride in the polyelectrolyte PAH main chain, generates secondary amine, It is electrically charged under extensive pH sets of conditions.But, such benzyl is modified the spatial volume added for amido, This can influence the interaction of charge-charge.In addition, being modified charged groups meeting with hydrophobic group in polyelectrolyte main chain Cause the reduction of charged groups number.For example, the amido of PAH results in acid amides company with the modification of benzyl chloride Connect, it is not charged groups, thus causes the reduction of charged groups number in main chain.Therefore, charged groups number Reduction can also influence the ability that the solubility and polymer of polymer are combined by charge-charge interaction.
When some polymer of the present invention are cation and other are anion, the polymerization of hydridization can be also synthesized Thing, it includes polyelectrolyte main chain, and the main chain is cation, and is entered with one or more hydrophobicitys and anionic group Modification is gone.In the case of such hybridized polymer, unmodified group is complexed for being formed on cationic polyelectrolyte The being in response to property of salt of thing, and the anion-modified group on main chain can combine the biology with net positive charge of interest point Son.Therefore, unmodified cation group in the polyelectrolyte main chain and the ratio of anion and hydrophobic group are for true It is important for the required solubility and stimulation of the fixed polymer complex and capture biomolecule of interest.For example, should On polyelectrolyte main chain very few unmodified cation group can cause to stimulate it is limited arrive without response.And on the contrary, Very few anionic group can limit the ability for capturing biomolecule of interest.
Amount necessary to hydrophobic group is modified polyelectrolyte main chain and the type and amount of stimulation used can be based on Using the biomolecule of interest and condition used of polymer purification, and the intrinsic solubility of the main polymer chain and point Son is measured to determine.For example in order to stimulate the consumption of such as multivalent salts minimum, it is therefore desirable to be to have to be connected to the poly- electricity Solve more hydrophobic groups on matter main chain.Selectable, the hydrophobicity needed for the amount that improving multivalent ion stimulates is reduced changes Property degree or hydrophobic modified percentage.In some cases, if such as multivalent ion is stimulated in very high concentration, It can be completely eliminated for the need for hydrophobic modified.It is selectable, improve polymer molecular weight and reduce polyelectrolyte main chain Intrinsic solubility, and low (5% or lower) polyelectrolyte can be allowed hydrophobic modified or without poly- electrolysis Matter is hydrophobic modified.But, hydrophobic modified cancellation can be improved in high dose of polymer, for low molecule amount or The residual polymerization object amount of bigger dissolubility main polymer chain.
In different embodiments, the hydrophobic modified degree on polyelectrolyte main chain is 1%-85% or 5%- 50%.Therefore, the biomolecule of interest combined depending on stimulating responsive polymer, hydrophobic modified percentage is at least 1%th, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%th, 70%, 75%, 80% or 85%.
In some embodiments, it is used as stimulation using the salt for forming complex compound.In different embodiments, network is formed The concentration of the salt of compound is 2mM-500mM, or 25mM-100mM.The salt of exemplary formation complex compound includes but is not limited to many Valency ion such as such as citrate, phosphate, sulfate and EDTA, and ion correlation salt such as perchlorate, dodecane Base sodium sulfate salt, dodecyl phenylsulfate, the chloro- 2- nitrophenols anion of Fe (II) -4-, tetraphenylboronic acid sodium salt and hexanitro Diphenol amine (see, for example, ANALYTICAL SCIENCES, in December, 1987, volume 3 page 479).Generally, people in the art Member is familiar with the salt of numerous formation complex compounds, and it is well known in the prior art, and may be used as polymer described herein Stimulation.
For cause precipitation needed for formation complex compound salt amount depend on factor such as such as pH, polymer concentration and The concentration of biomolecule of interest in the sample.For example, some polyelectrolyte such as PAH has as pH changes The charge density degree of protonation (amine).With pH increase, the reduction of charge density level so causes the thorn needed for precipitation Sharp degree will differ from the degree in relatively low pH or higher charge density state.
Generally, stimulating responsive polymer of the invention can be added to the feed (feedstock) containing target molecule Or in the sample containing target molecule, it is solid form or liquid form.Final polymer concentration is typically 0.01%-2%.In certain methods described herein, polymer and the mixture of biomolecule of interest are produced, is then added Entering stimulates the salt such as multivalent anions for for example forming complex compound.The amount of stimulation can depend on polymer concentration.Such as 2% Polymer concentration will need the stimulation of higher amount to cause polymer to precipitate.It is important that by the stimulation with appropriate or slightly mistake The amount of amount is applied, it is therefore an objective to ensure the response by the stimulation to carry out the complete precipitation of polymer.This and polymer flocculation Change forms contrast, wherein too high feeding coal result in problematic residual polymer.
The present invention can be used in a variety of purification schemes.The stimulating responsive polymer can methods described any step Suddenly use, although preferred use be in purge process or capture target molecule during, make when methods described starts With.Single stimulating responsive polymer or the mixture of polymer can be added in one or more steps, then be made Stimulated to precipitate with one or more.The stimulation can be (that is, one or more miscellaneous in polymer and the biomolecule of interest Matter or desired target molecule) combine before, among or apply afterwards.Equally, the stimulation can remove sediment (its Typically solid form) before, among or it is after-applied.Sediment can then use one kind or many known in the art The technology of kind and/or these such as such as filtering, sedimentation, centrifugation or any other solid/liquid separation sides described here Method or simultaneously, the combination of the method for parallel or continuous separation scheme removes.
The addition of the stimulating responsive polymer can be completed by several ways.Adding stimulating responsive polymer Before, cell culture medium can be adjusted to desired condition, such as adjustment (for example reduces) pH and/or conductivity.The stimulation Then responsive polymer can be added in cell culture medium, and mix.The stimulating responsive polymer can be with liquid Or solid form is added.Solution containing polymer can be prepared correctly in itself, to cause it by the pH of cell culture medium It is adjusted to desired condition.For example, the stimulating responsive polymer is soluble in spirit acid solution.This acetic acid solution Concentration can be changed based on volume, fermentation liquid condition and protein concentration, it is therefore an objective to be gathered by adding the stimulating responsive Thus compound produces desired polymer concentration and pH value of solution come pH adjustment necessary to providing.The stimulating responsive polymer Can be to occur the addition of the concentration of spontaneous flocculating, typical concentration is 0.01-0.1%wt polymer or 0.01-0.5%wt Polymer, this depends on type and percentage of solids, so that the solution becomes blushing and initially forms sediment.It may be selected , not occur spontaneous flocculating the dense of the combination of Polymer-bioactive molecule can still occur for the stimulating responsive polymer Degree is added, such as typical concentration is 0.5%-2%wt polymer, and the solution can than it is initial when solution it is transparent or Slightly blushing or more muddy.Equally, the stimulating responsive polymer can with occur the spontaneous flocculating of mixture and polymer- The concentration of the combination of biomolecule is added.
Although in purification schemes using stimulate be more it is desirable that because it alleviate and polymer excessively feed phase The problem of some problems closed are for example in flocculating method, it is contemplated that polymer described here can also be used Make flocculant.
In a kind of exemplary purification schemes, the stimulating responsive polymer of the present invention is added after the fermentation was completed Into cell culture, and by polymer formulation come combine be not desired target molecule biomolecule of interest. In such method, stimulating responsive polymer is added in cell culture under the first set of conditions, the condition for example may be used Be before combining the addition of polymer of biomolecule of interest, among or the condition that is adjusted afterwards.At this After stimulating responsive polymer is added under the first set of conditions, added stimulating under a second set of conditions, thus produce and include Biomolecule (such as one or more impurity such as cell, cell fragment, host cell proteins matter, DNA, endogenous toxic materials of interest Element and virus) sediment.Thus the solid sediment then can produce clarification by centrifuging and/or being filtered to remove The cell culture fluid crossed.The cell culture fluid of the clarification formed then can by using chromatographic media capture step, To combine desired target molecule.The target molecule can then be eluted from capture step.Therefore in some cases, pass through Using the stimulating responsive polymer of the invention of one or more impurity can be removed in clarification steps, it can reduce/eliminate Or change the quantity of other step.
In some embodiments, stimulating responsive polymer be the polymer combine not be being closed for target molecule The biomolecule of note it is such under the conditions of be added in cell culture.In the stimulating responsive polymer in desired solution After sufficient condition mixing, the flocculate for including biomolecule of interest is formed.Removed by primary clarification containing of interest Biomolecule solid.The cell culture fluid formed is collected, and is administered to stimulating on the residual polymer of precipitation. The residual polymer of the precipitation is then removed by secondary clearing.By the cell culture solution of the clarification formed by using The capture step of chromatographic media carrys out combining target molecule.Can be in any purification step after primary clarification by stimulating Addition remove residual polymer.It is also possible to remove residual polymer in any step addition stimulation or whole Repeatedly addition is one or more in method stimulates.
In another purification schemes, after the fermentation has been completed, stimulating responsive polymer is combined suitable for polymer It is added under conditions of one or more impurity in cell culture fluid, to cause the polymer not combining target molecule.In the thorn After sharp responsive polymer is sufficiently mixed under desired solution condition, adding is stimulated, and it is formed with one or more impurity Solid sediment.The solid sediment is by centrifuging and/or being filtered to remove.The cell culture of the clarification formed is molten Liquid is by that can combine the filter of the stimulating responsive polymer.Those skilled in the art can be readily selected/determine this Sample can combine the stimulating responsive polymer filter.For example, such filter can be provided, it has and its dozen Calculate the similar performance of the biomolecule of interest combined.It is selectable, such filter can be used, it has with being closed The charged groups of the biomolecule identical charges of note and the charged groups with stimulating responsive polymer opposite charges.
Film, packed bed or filter can be used for removing polymer from solution.Such as the moon containing sulfonic acid group Ionic membrane can be used for removing polyamine stimulating responsive polymer.It is also possible to using have with for precipitating the polymer The similar binding ability of stimulation filter.If the stimulation is multivalent anions, offer surface is contained can be from solution The filter of the middle multivalent anions for removing the polymer.For example, with polyvinyl phosphoric acid salt be modified film can with can It is complexed the closely similar mode of phosphate anion of polyamine to combine polyamine, thus causes precipitation.It is also possible to poly- using using The pearl (such as polymethacrylates) that vinyl phosphoric acid salt is modified.In some embodiments, this can be removed in use After the filter of stimulating responsive polymer is filtered to solution, by the solution formed by using chromatographic media Capture step carrys out combining target biomolecule.Chromatographic media, deep bed filter or other porous material (its energy can be used Enough combine the stimulating responsive polymer) remove polymer.Equally can be with adsorptivity device is in single step or is more than The polymer is removed in one or more step.Furthermore, it is possible to be added to mixture in the polymer via adsorptivity device In after any step in remove the polymer.
It can be expected that the present invention can be used in a variety of changes of purification schemes described herein.The stimulating responsive Polymer may be substituted for or strengthen clarification steps and capture both steps.It is, for example, possible to use two kinds individually stimulate Responsive polymer;First polymer combines one or more impurity but not combining target molecule, second polymer combination mesh Mark molecule.Both polymer can be used the step of respectively or in single step.It is also possible to use single polymerization Thing, it has the functional group for being capable of combining target molecule and can combine the polyelectrolyte main chain of one or more impurity.The list One polymer can in single step or multiple steps combining target molecule and one or more impurity.It is different at several Precipitation/stimulation addition or cleaning step after, can then carry out target molecule from the elution in the polymer.The stimulation (it is going out by virus to the solid and impurity that responsive polymer can be added after capture step and suspended for clarification Life birth life), or added after other steps after capture step.The stimulating responsive polymer can also replace or Refined (polishing) step of person's enhancing.
In some embodiments, including viral inaction steps (that is, by solution be exposed to low pH, surfactant or Heating).The solution condition can be adjusted, and by a series of purification step, (that is, one or more ions is handed over by sample Change, hydrophobic interaction, mixed mode and other) be processed.Then the solution can undergo a series of filtration step, Including virus filtration and ultrafiltration or diafiltration.
In order to be easier to understand the present invention, some terms are defined first.Other is defined in whole detailed description Illustrate.
I.Definition
As what is be used interchangeably herein, term " stimulation " represents the physics or chemical change in environment, and which results in this The response of the stimulating responsive polymer of invention.Therefore, the present invention provides new polymer, its for stimulating being in response to property, And the stimulation result in the change of the polymer solubility.The stimulation that one or more polymer described herein are responsed to which Example include but is not limited to such as temperature change, conductance variation and/or pH change.In some embodiments, bag is stimulated Include and the salt of complexing agent or formation complex compound is added in sample.In multiple embodiments, it is typically in polymer to stimulate Added after being added in sample.Although the stimulation can also among polymer is added to sample or before add.
It is used as term " polymer " used herein " refer to being covalently attached institute by two or more monomeric unit The molecule of formation.These monomeric units can be synthesized or be naturally occurring.This formed by repeat unit gathers Compound can be straight chain or branched.The example of polymer include but is not limited to polyethylene glycol, polypropylene glycol, polyethylene, PAH, polyvinyl alcohol, polystyrene and copolymer (such as polystyrene-co-poly pyridine, polyacrylic acid-copolymerization- Methyl methacrylate, Pu Lunuonike (pluronics), PF68 etc.).In some embodiments of the present invention, polymer Include polyelectrolyte main chain.Equally described here is copolymer, and it can be used in the method for the present invention, wherein the copolymer It is stimulating responsive.Generally, it should be understood that in the case of polymer, monomeric unit is same type, and copolymer is logical Often there is different types of monomeric unit.
It is polymer or copolymer as term used herein " stimulating responsive polymer ", it is adding stimulation The performance physically and/or chemically changed is shown afterwards.Typical stimuli responsive is the change of polymer solubility.Such as polymer NIPA is water miscible when temperature is less than 35 DEG C, but becomes insoluble in about 35 DEG C of temperature In water.In a kind of specific embodiment, stimulating responsive polymer is PAH or polyvinylamine polymerization Thing, it stimulates (such as phosphate radical stimulation) being in response to property for multivalent ion.
Containing comprising two or more monomeric unit is referred to as term used herein " polyelectrolyte main chain " Carbon polymer, the unit of wherein at least 50% or at least 55% unit or at least 60% unit or at least 65% unit or at least 70% unit or at least 75% unit or at least 80% unit or at least 85% unit or at least 90% unit or at least 95% unit, include electrically charged functionality.In other words, At least 50% monomeric unit includes charged groups, and which form a part for the unit.In some embodiments, here Described polyelectrolyte main chain includes at least two or more monomeric units, wherein each unit includes electrically charged function Property.In the case of the polyelectrolyte main chain of polymer, wherein each monomeric unit includes electrically charged functionality, it is such poly- Compound can be referred to as " continuous polyelectrolyte ".Exemplary polyelectrolyte matter includes but is not limited to PAH, polyvinylamine, gathered Acrylic acid, polyethyleneimine, chitosan and polyvinyl phosphoric acid.It can also be expected that (it is different from one or more materials The monomeric unit) it may be coupled on polyelectrolyte main chain.
Apolar substance or chemical group are referred to as term used herein " hydrophobic group ", its parent to water Conjunction property is very low to not having.Exemplary hydrophobic group includes but is not limited to phenyl, the tert-butyl group, cyclic hydrocarbon, multicyclic aliphatic hydrocarbon, many PAH and short hydrocarbon such as hexyl and octyl group.In a kind of specific embodiment, the hydrophobic group is phenyl.This is hydrophobic Property group can also be nonhydrocarbon, and comprising hetero atom such as nitrogen, oxygen, sulphur, phosphorus.In the various embodiments of the present invention, There is provided stimulating responsive polymer, it includes polyelectrolyte main chain, and the main chain has the charged groups being connected in main chain On one or more hydrophobic groups.It is not intended to be limited to theory, it should be understood that be connected to hydrophobic on polyelectrolyte main chain Property group number be important for changing polymer solubility thus to improve the stimulating responsive of the polymer.But, no Desirably there are many hydrophobic groups, it is insoluble that it imparts the polymer water when not stimulating.
Percentage of the charged groups in polyelectrolyte main chain (it is modified with hydrophobic group) is commonly referred to as this " the hydrophobic modified percentage " of polymer.Therefore in various embodiments, hydrophobic modified percentage is important, and It is 1%-85% or 5%-85%.Therefore hydrophobic modified percentage can be at least 1% or at least 2% or at least 3% or at least 4% or at least 5% or at least 6% or at least 7% or at least 8% or at least 9%, Or at least 10% or at least 15% or at least 20% or at least 25% or at least 30% or at least 35% or at least 40% or at least 45% or at least 50% or at least 55% or at least 60% or extremely Few 65% or at least 70% or at least 75% or at least 80% or at least 85%.
It is electrically charged that unmodified polyelectrolyte is commonly referred to as term used herein " hydrophobic modified percentage " The ratio for the polyelectrolyte charged groups that group and hydrophobic group are modified, as total polyelectrolyte monomeric unit in poly- electrolysis Percentage in matter main polymer chain.
In some embodiments, the hydrophobic group being connected on polyelectrolyte main chain is further hydrophobic with being connected to Charged groups on property group, it is significantly different from the material of the charged groups in main chain.
Straight chain is commonly referred to as term used herein " alkyl " or branched hydrocarbon chain.Straight chain or branched chain hydrocarbon Chain refers to any substituted or unsubstituted comprising non-cyclic carbon compound, including such as alkane, alkene and alkynes.Alkane The example of base includes low alkyl group such as methyl, ethyl, n-propyl, isopropyl, normal-butyl, isobutyl group, the tert-butyl group or dissident Base;Senior alkyl is such as n-heptyl, n-octyl, iso-octyl, nonyl, decyl;Low-grade alkenyl such as vinyl, acrylic, third Alkynyl, cyclobutenyl, butadienyl, pentenyl, n-hexylene base or isohexenyl;With senior alkenyl such as nhepene base, just pungent Alkenyl, isooctene base, nonenyl, decene base etc..Numerous straight chain (that is, branched) familiar to the person skilled in the art and branched Alkyl, it is included in the invention.
In addition, such alkyl can also include different substituents, wherein one or more hydrogen atoms are by institute of functional group Substitute.The example of functional group includes but is not limited to carboxylic acid group, sulfonic group, phosphate etc..It is used as term used herein " alkenyl " Refer to straight chain or branched hydrocarbon chain, the connection of wherein at least one carbon-to-carbon is carbon-to-carbon double bond.
The alkyl that end is replaced by least one aryl is referred to as term used herein " aralkyl ".
The alkenyl that end is replaced by least one aryl is referred to as term used herein " aryl alkenyl ".
The hydrocarbon ring with conjugate double bond system is referred to as term used herein " aryl ", often comprising at least six N (pi) electronics.The example of aryl includes but is not limited to phenyl, naphthyl, anisyl, toluyl groups and xylyl.
Straight chain is referred to as term used herein " fluorocarbons " or branched carbochain, wherein one or more Hydrogen atom is replaced by fluorin radical.The fluorocarbons chain of straight chain or branched chain is commonly referred to as any substituted or unsubstituted Contain acyclic carbon compound, including such as alkane, alkene and alkynes.The example of alkyl include low alkyl group such as methyl, Ethyl, n-propyl, isopropyl, normal-butyl, isobutyl group, the tert-butyl group or isohesyl;Senior alkyl for example n-heptyl, n-octyl, Iso-octyl, nonyl, decyl etc.;Low-grade alkenyl such as vinyl, acrylic, propinyl, cyclobutenyl, butadienyl, pentenyl, just Hexenyl or isohexenyl;With senior alkenyl such as nhepene base, positive octenyl, isooctene base, nonenyl, decene base. (that is, straight chain) and branched alkyl generally it should be understood that numerous straight chain familiar to the person skilled in the art, it is in this hair In bright scope.In addition, such alkyl can also include a variety of substituents, wherein one or more hydrogen or one or many Individual fluorine atom is substituted by functional group.The example of functional group includes but is not limited to carboxylic acid group, sulfonic group, phosphate etc..
It is the group that other functionality is assigned for polymer described herein as term used herein " functional group ". In other words, functional group is such group, and it is different from hydrophobic group, and is also connected to example on the polyelectrolyte main chain Such as it is connected in the charged groups of main chain.For example in some embodiments, functional group can be used to change the polymer Binding ability part, e.g. carboxylic acid, sulfonic acid, sulfate, primary amine, quaternary amine and diethylamino.The functional group can be with Change the performance of polymer or other desired performance is provided, for example, such as change stimulating responsive or cause the polymerization Thing response second is stimulated.Include but is not limited to hydroxy-acid group (pH responses for changing the exemplary functional groups of stimuli responsive behavior Property), pyridine groups (pH responses) and NIPA group (temperature-responsive).
It is commonly referred to as providing the specific binding energy with another material as term used herein " part " The material of power.The example of " part " includes but is not limited to ion-exchange group, biological affinity or biologic specificity group, dredged Aqueous group, thiophilic property interaction group, chelate or chelation group, with target compound there is so-called pi-pi phases Group, H-bonding groups and the hydrophilic radical of interaction.
Refer to adding flocculant such as polymer described herein to solution as term used herein " flocculating " In, to remove the insoluble or soluble impurity of one or more suspensions.The polymer must be added to such concentration In the solution, i.e. the concentration allows to spontaneously form insoluble aggregates, it can be via typical solid- liquid separation method from molten Removed in liquid.
Refer to intending with one kind described herein as term used herein " composition ", " solution " or " sample " Or the target molecule that is purified of a variety of stimulating responsive polymers or desired product and one or more undesirable The mixture of material or impurity.In some embodiments, the sample has target molecule or desired comprising wherein secretion The feed or cell culture medium of product.In some embodiments, the sample includes target molecule (such as therapeutic protein Or antibody) and one or more impurity (for example host cell proteins matter, DNA, RNA, lipid, cell culture additive, Cell and cell fragment).In some embodiments, the sample includes target molecule of interest, and it is secreted in cell culture In base.In some embodiments, it is intended to purified therefrom with one or more stimulating responsive polymers described herein The sample of target molecule is " partially purified " before the sample is contacted with stimulating responsive polymer.Partial purification can example Such as by cause the sample carry out one or more for example such as one or more non-affinity chromatography steps of purification step come Complete.The target molecule can be from one or more undesirable materials or impurity by precipitating one or more impurity Or separated by precipitating the target molecule.
In some embodiments, stimulating responsive polymer of the invention combines of interest under the first set of conditions Biomolecule, itself is target molecule or product (such as target protein or polypeptide), and under a second set of conditions The target molecule is precipitated, for example, is precipitated by adding stimulation into the sample.In other embodiments, biology of interest Molecule is the molecule of non-target molecule.In other words, the of interest of stimulating responsive polymer combination described herein is passed through Biomolecule can be such molecule, and it does not expect to be combined with the target molecule in sample.It is not intended to be limited to theory, It is expected that in some embodiments, stimulating responsive polymer of the invention is combined when adding and stimulating and to precipitate host thin One or more in born of the same parents' protein, DNA, full cell, cell fragment, virus, endotoxin and/or cell culture additive. Therefore target molecule (such as target protein or polypeptide) can use polymer described herein, by precipitating desired mesh Mark molecule is purified, described impurity by precipitating one or more undesirable materials (such as one or more impurity) It may reside in the sample containing desired target molecule.
As term used herein " precipitation ", " precipitability " or " precipitation " refer to combine (for example with it is of interest Biomolecule complexing) or uncombined polymer become water-free and/or insoluble from aqueous and/or dissolved state State.
Referred to as term used herein " biomolecule of interest " poly- by stimulating responsive described herein Compound is come any molecule for combining and precipitating.For example, the biomolecule of interest can be that desired target molecule is for example all Product as desired either polypeptide (such as antibody) or it can be undesirable material, it is needed from containing desired mesh Mark in the sample of molecule and remove.Such undesirable material include but is not limited to be selected from host cell proteins matter, DNA, One or more in RNA, protein aggregate, cell culture additive, virus, endotoxin, full cell and cell fragment Impurity.
As what is be used interchangeably herein, term " target molecule ", " target biological molecules ", " desired target molecule " and " desired target biological molecules " are commonly referred to as polypeptide or product of interest, it may be desirable to by its from it is one or more not In for example one or more impurity (it may reside in the sample containing polypeptide or product of interest) of desired material Isolated or purified.As the term " protein of interest " being used interchangeably herein, " target polypeptides ", " polypeptide of interest " and " target protein " is commonly referred to as the stimuli responsive of therapeutic protein or polypeptide, including but not limited to the plan present invention The antibody that property polymer is purified.
It is commonly referred to as having as the term " polypeptide " being used interchangeably herein or " protein " and is more than about 10 ammonia The peptide and protein of base acid.In some embodiments, stimulating responsive polymer described herein be used for from protein Or polypeptide is present in sample and separates one or more undesirable materials and the protein or polypeptide together.At some In embodiment, one or more materials are one or more impurity, and it can be with protein or polypeptide one to be purified Rise and be present in sample.As described above, in some embodiments, being stimulated by being added to sample, stimulation described herein rings Answering property polymer specifically binds and precipitated protein or polypeptide of interest.In other embodiments, by adding Stimulate, stimulating responsive polymer described herein combines and precipitated such material, and it is different from protein of interest Or polypeptide such as such as host cell proteins matter, DNA, virus, full cell, cell fragment and cell culture additive.
In some embodiments, it is intended to the protein or many purified with stimulating responsive polymer described herein Peptide is mammalian proteins such as therapeutic protein or the protein that can be used in treatment.Exemplary protein includes But it is not limited to such as feritin;Growth hormone, including human growth hormone and bovine growth hormone;Somatotropin releasing factor;First shape Other glandular hormone;Thyroid-stimulating hormone (TSH);Lipoprotein;α -1- antitrypsins;INSULIN A-chain;Insulin B-chain;Proinsulin; Follicle-stimulating hormone (FSH);Calcitonin;Luteinising hormone;Hyperglycemic factor;Clotting factor such as Factor IX C, factors IX, organize because Son and the von Willebrands factors;Anticoagulin such as protein C;Atrial natriuretic peptide;Pulmonary surfactant;Plasminogen is lived The plasminogen activator (t-PA) of agent such as urokinase or human urine or organization type;Bombesin;Fibrin ferment;Hematopoiesis is given birth to The long factor;The knurl subcutaneous ulcer factor-α and-β;Enkephalinase;RANTES (expression and secretion of the common T- cells of control activation);People is huge Phagocyte inflammation protein (MIP-1- α);Haemocyanin such as human albumin;Muellerian inhibitory substances;Relaxin A- chains;Relaxin B- chains;prorelaxin;The related peptide of mouse promoting sexual gland hormone;Microprotein such as beta-lactamase; Deoxyribonuclease;IgE;The related antigen (CTLA) of cytotoxin T- lymph corpuscles, such as CTLA-4;Inhibin;Activin; The Vascular endothelial growth factor (VEGF);The acceptor of hormone or growth factor;A-protein or D;Rheumatoid factor;Close nerve The factor for example bone source close neural factor (BDNF), NT-3, -4, -5, or -6 (NT-3, NT-4, NT-5 or Person NT-6), or nerve growth factor such as NGF- β;The growth factor (PDGF) in blood platelet source;Fibroblast growth because Son such as α-FGF and β-FGF;EGF (EGF);TGF (TGF) such as TGF- α and TGF-β, including TGF-β 1, TGF-β 2, TGF-β 3, TGF β 4 or TGF-β 5;Insulin like growth factor-1 and-II (IGF-I and IGF-II); Des (1-3)-IGF-I (brain IGF-I), insulin-like growth factor binding proteins (IGFBP);CD protein such as CD3, CD4, CD8, CD19, CD20, CD34 and CD40;Erythropoietin(EPO);The bone growth inducing factor;Antitoxin;Bone morphogenic protein Matter (BMP);Interferon such as interferon-' alpha ' ,-β and-γ;Flora stimulating factor (CSF) such as M-CSF, GM-CSF and G-CSF; Interleukins (Il) such as IL-1 to IL-10;Superoxide dismutase;T-cell receptors;Surface membrane protein matter;Decay accelerates The factor;Viral antigen (viral antigen) such as such as AIDS coatings of a part;Transport protein;Homing receptor;Address Element;Regulatory protein matter;Integrin such as CD11a, CD11b, CD11c, CD18, ICAM, VLA-4 and VCAM;Tumour correlation Antigen such as HER2, HER3 or HER4 acceptor;With the fragment and/or variant of any polypeptide listed above.
In addition, in some embodiments, the protein or polypeptide purified using the thorn photosensitive polymerization thing of the present invention is anti- Body, its function fragment or variant.In some embodiments, protein of interest is recombinant cell protein matter, and it contains The Fc regions of immunoglobulin.
Term " immunoglobulin ", " Ig " or " antibody " (in this used interchangeably) refers to having by two heavy chains and two The protein of basic four polypeptide chain structures of individual light chain composition, described chain is come stable for example by the disulfide bond of interchain , it being capable of molecule of the antigen binding.What term " single-chain immunoglobulins " or " single-chain antibody " (in this used interchangeably) referred to It is the protein with two polypeptide chain structures being made up of a heavy chain and a light chain, described chain is for example by interchain Peptide bond come what is stablized, it being capable of molecule of the antigen binding.Term " domain " refers to the spherical region containing heavy chain or light chain polypeptide Domain, described polypeptide is included for example by beta-pleated sheet and/or the stable peptide ring of interchain disulfide bond (such as comprising 3-4 peptide Ring).Domain is further referred to as " constant " or " variable " herein, and it is based in the case of " constant " domain different types of In the domain of member with respect to lack order change, or in the case of " variable " domain in the domain of different types of member substantially Change.Antibody or polypeptide " domain " are often interchangeably referred to as antibody or polypeptide " region " in the art.Antibody light chain " constant " domain is interchangeably referred to as " chain constant region ", " light-chain constant domains ", " CL " region or " CL " domain.Heavy chain of antibody " constant " domain is interchangeably referred to as " heavy chain constant region ", " heavy-chain constant domains ", " CH " region or " CH " domain.Antibody light chain " variable " domain is interchangeably referred to as " light chain variable region ", " light-chain variable domain ", " VL " region or " VL " domain.Heavy chain of antibody " variable " domain is interchangeably referred to as " heavy chain variable domain ", " heavy chain variable domain ", " VH " region or " VH " domain.
Immunoglobulin or antibody can be monoclonals or polyclonal, and can be with monomer or polymer Form is present, for example, exist with the IgM antibody of pentamer form presence and/or with monomer, dimer or multimeric forms IgA antibody.Immunoglobulin or antibody can also include multi-specificity antibody (such as bispecific antibody) and antibody fragments, As long as they keep or are modified to include part-specific binding domains.Term " fragment " refers to antibody or antibody chain A part, its include the amino acid less than complete or complete antibody chain.Fragment can be via complete or complete The processing of the chemistry or enzyme of complete antibody or antibody chain is obtained.Fragment can also be obtained by recombinant cell means.When When recombinant cell is produced, a part for the bigger protein that fragment can be using single expression or as referred to as fused protein is come Expression.Exemplary fragment include Fab, Fab ', F (ab ') 2, Fc and/or Fv fragments.Exemplary fused protein includes Fc Fused protein.
Generally, immunoglobulin or antibody are related to " antigen " of interest.It is preferred that, antigen is important many on biology Peptide, and the administration that antibody is the mammal by disease or disorder can be produced into the treatment benefit for mammal. But, antibody (such as tumour relevant glycolipid antigen relevant with non-polypeptide antigen;Referring to United States Patent (USP) No.5091178) It is also what is be expected.In the case of antigen is polypeptide, it can be that transmembrane molecule (such as acceptor) or part are for example given birth to The long factor.
Refer to being obtained from the antibody of basic comparator antibody group as term used herein " monoclonal antibody ", i.e. described The single antibody that includes is identical in group, except the mutation of naturally occurring that there may be on a small quantity.Monoclonal antibody is height It is specific, it is related to single antigenic site.In addition, with conventional (polyclonal) antibody preparation, (it, which is typically include, is related to The different antibody of different factor of determinations (epitope)) on the contrary, each monoclonal antibody be related to it is single on antigen Factor of determination.Qualifier " monoclonal " represents the characteristic of the antibody obtained from essentially identical antibody population, and is not construed as leading to The production for the antibody crossed needed for any special method.For example, the monoclonal antibody used in the present invention can pass through Kohler etc. People, Nature 256:The hybrid cell that 495 (1975) are described first is manufactured, or can pass through recombinant cell DNA methods To manufacture (see, for example, United States Patent (USP) No.4816567)." monoclonal antibody " can also use such as Clackson et al., Nature 352:624-628 (1991) and Marks et al., J.Mol.Biol.222:Technology described in 581-597 (1991) is come Separated from antibiotic antibody library.
Monoclonal antibody can comprise additionally in " mosaic type " antibody (immunoglobulin), the weight of a portion and/or light Chain be with from predetermined substance or belong in the antibody of specific antibody type or subspecies corresponding sequence identical or Person is similar, and remaining chain is and from another material or belongs to the anti-of another antibody type or subspecies Corresponding sequence identical or similar in body and such antibody fragments, as long as they show desired bioactivity (United States Patent (USP) No.4816567;With Morrison et al., Proc.Natl.Acad.Sci.USA 81:6851-6855 (1984))。
When for herein when, term " hypervariable region " refers to the amino acid residue of antibody, and it is the original for causing antigen binding Cause.The hypervariable region includes amino acid residue, and it comes from " complementarity determining region " or " CDR " (that is, residue in light-chain variable domain 31-35 (H1), 50-65 (H2) and 95-102 in 24-34 (L1), 50-56 (L2) and 89-97 (L3) and heavy chain variable domain (H3);Kabat et al., Sequences of Proteins of Immunological Interest, the 5th edition, Public Health Service, National Institutes of Health, Bethesda, Md. (1991)) and/or from " high Become ring (hypervariable loop) " (residue 26-32 (L1), 50-52 (L2) and 91-96 (L3) i.e. in light-chain variable domain With the 26-32 (H1) in heavy chain variable domain, 53-55 (H2) and 96-101 (H3);Chothia and Lesk J.Mol.Biol.196: 901-917 (1987)) these residues." framework " or " FR " residue is these variable domain residues, rather than defined here Some hypervariable region residues.
Non-human (such as Muridae) antibody of " humanization " form is Chimeric antibodies, and it, which is included, comes from inhuman para-immunity The minmal sequence of globulin.In greatly degree, humanization antibody is human immunoglobulin (acceptance antibody), wherein by The some hypervariable region residues of body are by non-human material's (donor antibody) with desired specificity, affinity and ability from below Some hypervariable region residues replaced:Such as mouse, rat, rabbit or inhuman primate.In some cases, the mankind Fv frame areas (FR) residue of immunoglobulin is replaced by corresponding non-human residues.In addition, humanization antibody can be included The residue not having in receptor antibody or donor antibody.These can be carried out to be modified further to improve antibody performance.Generally, The humanization antibody include it is substantially all at least one and typical two kinds of variable domains, wherein whole or substantially Whole hypervariable rings corresponds to these of non-human immunoglobulin, and whole or substantially all of FR regions are Human immunoglobulin sequence these.The humanization antibody optionally will also be constant comprising at least one of immunoglobulin Region (Fc), typically human immunoglobulin.Further details are referring to Jones et al., Nature 321:522-525 (1986);Riechmann et al., Nature 332:323-329(1988);And Presta, Curr.Op.Struct.Biol.2: 593-596(1992)。
In some embodiments, it is using the stimulating responsive polymer antibody that is separated or purified of the present invention Therapeutic antibodies.Exemplary treatment antibody includes such as Trastuzumab (trastuzumab) (HERCEPTINTM, Genentech, Inc., Carter et al. (1992) Proc.Natl.Acad.Sci.USA, 89:4285-4289;United States Patent (USP) No.5725856); Anti-CD 20 antibodies such as mosaic type anti-CD 20 " C2B8 " United States Patent (USP) No.5736137);rituximab(RITUXANTM), Ocrelizumab, the mosaic type or humanization variant (United States Patent (USP) No.5721108 of a kind of 2H7 antibody;WO04/056312) Or tositumomab (BEXXAR.TM);Anti- IL-8 (St John et al. (1993) Chest, 103:932, and WO95/ 23865);Anti-VEGF antibodies, including humanization and/or the sexually matured anti-VEGF antibodies of affinity such as humanization anti-VEGF resist Body huA4.6.1bevacizumab (AVASTINTM, Genentech, Inc., (1992) growth factor 7 such as Kim:53-64, WO96/30046, WO98/45331);Anti- psca antibody (WO01/40309);Anti-CD 40 antibodies, including S2C6 and its humanization Variant (WO00/75348);Anti- CD11a (United States Patent (USP) No.5622700;WO98/23761;Steppe etc. (1991) Transplant Intl.4:3-7;Hourmant etc. (1994) Transplantation 58:377-380);It is anti-IgE (Presta etc. (1993) J.Immunol.151:2623-2632;WO95/19181);Anti- CD18 (United States Patent (USP)s No.5622700;WO97/26912);It is anti-IgE, including E25, E26 and E27 (United States Patent (USP) No.5714338;United States Patent (USP) No.5091313;WO93/04173;U.S. No.5714338);Anti- Apo-2 receptor antibodies (WO98/51793);Anti-TNF-α resists Body, including cA2 (REMICADETM), CDP571 and MAK-195 (United States Patent (USP) No.5672347;Lorenz etc. (1996) J.Immunol.156(4):1646-1653;Dhainaut etc. (1995) Crit.Care Med.23 (9):1461-1469);It is anti- Tissue Factor(TF)(EP0420937B1);The integrins (WO98/06248) of 4 β of Anti-Human's class α 7;Anti-EGFR, it is chimeric Or the antibody of humanization 225 (WO96/40210);Anti-CD 3 antibodies such as OKT3 (United States Patent (USP) No.4515893);Anti- CD25 Or anti-tac antibody such as CHI-621SIMULECTTMAnd ZENAPAXTM(United States Patent (USP) No.5693762);Anti-CD 4 antibodies Such as cM-7412 antibody ((1996) Arthritis Rheum39 (1) Choy:52-56);Anti-CD 52 antibody is for example CAMPATH-1H (Riechmann etc. (1988) Nature332:323-337);Anti-FC receptor antibodies such as M22 antibody is related to (1995) J.Immunol.155 (10) such as Fc γ RI such as Graziano:4996-5002;Anti- carcinomebryonic antigen (CEA) antibody is for example HMN-14 (Sharkey etc. (1995) Cancer Res.55 (23Suppl):5935s-5945s;It is related to the anti-of breast epithelial cells Body, including huBrE-3, hu-Mc3 and CHL6 (Ceriani etc. (1995) Cancer Res.55 (23):5852s-5856s;With Richman etc. (1995) Cancer Res.55 (23Supp):5916s-5920s);It is attached to the antibody of colorectal cancer cells for example C242 (Litton etc. (1996) Eur J.Immunol.26 (1):1-9);Anti-cd 38 antibodies, such as AT13/5 (Ellis (1995)J.Immunol.155(2):925-937);Anti- CD33 antibody is such as Hu M195 ((1995) Cancer Jurcic Res 55(23Suppl):5908s-5910s and CMA-676 or CDP771;Anti- CD22 antibody such as LL2 or LymphoCide (Juweid etc. (1995) Cancer Res 55 (23Suppl):5899s-5907s);Anti- EpCAM antibody is for example 17-1A(PANOREXTM);Anti- GpIIb/IIIa antibody such as abciximab or c7E3Fab (REOPROTM);Anti- RSV resists Body such as MEDI-493 (SYNAGISTM);Anti- CMV antibody such as PROTOVIRTM);Anti-HIV antibody such as PRO542;Anti- liver For example anti-Hep B antibody OSTAVIR of scorching antibodyTM);Anti- CA125 antibody OvaRex;Anti- spy answers GD3 epitope antibody BEC2;The anti-antibody VITAXIN of α v β 3TM;Anti-Human's class kidney cell anticancrin such as ch-G250;ING-1;Anti-Human's class 17-1A Antibody (3622W94);Anti-Human's class colorectal tumours antibody (A33);It is related to the anti-human melanoma of GD3 gangliosides Antibody R24;Anti-Human's class squama cell carcinoma (SF-25);With anti-HLA (HLA) antibody such as Smart ID10 and Anti- HLA DR antibody Oncolym (Lym-1).
Referred to as the term " pollutant ", " impurity " and " fragment " being used interchangeably herein any external or disagreeable Material, including large biological molecule such as DNA, RNA, one or more host cell proteins matter (HCP or CHOP), endogenous toxic material Element, virus, lipid and one or more additives, it may reside in (for example resists containing protein or polypeptide of interest Body) sample in, the stimulating responsive polymer of protein or the polypeptide plan present invention is by itself and one or more External or disagreeable molecule separation.In some embodiments, stimulating responsive polymer described herein is from containing Protein or many of interest is combined and has precipitated in the sample of the protein or polypeptide of concern and one or more impurity Peptide.In other embodiments, stimulating responsive polymer described herein combines and has precipitated one or more impurity, thus Polypeptide or protein of interest are separated with one or more impurity.
Referred to as the term " Chinese hamster ovary cell protein " being used interchangeably herein and " CHOP " from China The mixture of the host cell proteins matter (" HCP ") of mouse ovary (" CHO ") cell culture.HCP or CHOP are usually as miscellaneous Matter be present in cell culture medium or lysate (for example containing protein or polypeptide of interest (for example antibody or The immunoadhesin expressed in Chinese hamster ovary celI) harvest cell culture fluid).Generally, CHOP is including protein of interest Mixture in amount provide for lipidated protein of interest measurement.Typically, CHOP is mixed in protein Amount in thing is to be represented relative to the number in parts per million of albumen quality of interest in mixture.
It should be understood that host cell is another mammalian cell types here, E.coli, yeast cells, insect is thin Born of the same parents or plant cell, HCP refer to the protein of the non-targeted protein present in the lysate of host cell.
Refer to that (for example nonprotein is added such molecule as term used herein " cell culture additive " Agent), it is added in cell culture processes to promote or improve cell culture or fermentation process.In some realities of the present invention Apply in scheme, stimulating responsive polymer described herein combines and precipitated one or more cell culture additives.Show Example property cell culture additive includes defoamer, antibiotic, dyestuff and nutrients.
Referred to as the term " number in parts per million " being used interchangeably herein or " ppm " for using described herein The amount of desired target molecule (such as target protein or antibody) purity that is purified of stimulating responsive polymer Degree.Therefore, it is this to measure the amount that can be used for measurement target molecule existing after purification process or for measuring not The amount of desired material.In some embodiments, unit " ppm " is used herein to represent impurity in solution, such as of interest The HCP or CHOP of protein (mg/ml) amount (unit is ng/ml) (that is, CHOP ppm=(CHOP ng/ml)/(closed The protein mg/ml of note).When the protein (such as by desivac) is dried, ppm refers to (CHOP ng)/(of interest Protein mg)).
Term " separation ", " purifying " and " separation " is exchanged for using stimulating responsive polymer described herein herein, Purification of target molecule (such as polypeptide of interest from composition or sample comprising target molecule and one or more impurity Or protein) context in.In some embodiments, the purity of target molecule in the sample is by using this place The stimulating responsive polymer stated removes (completely or partially) one or more impurity to improve from sample.Other one Plant in embodiment, the purity of target molecule in the sample is by the way that target molecule is sunk from one or more impurity of sample Form sediment what is come out and improve.
In some embodiments, purification process has been used one or more " chromatographic steps " in addition.Typically, if Need, these steps can use stimulating responsive polymer of the invention by one or more undesirable materials and target Molecule is separately carried out afterwards.
In some embodiments, institute is isolated, separates or purified using stimulating responsive polymer described herein The polypeptide of concern or " purification step " of protein can produce " uniform " or " pure " composition or sample Whole purification process a part, terminology used herein refers to composition or sample, and it is comprising of interest The HCP less than 100ppm is included in the composition of protein, it is selectable to be less than 90ppm, less than 80ppm, less than 70ppm, small In 60ppm, less than 50ppm, less than 40ppm, less than 30ppm, less than 20ppm, less than 10ppm, less than 5ppm or it is less than 3ppm HCP.
It is commonly referred to as term used herein " clarification (clarification) step " in purifying biomolecules One or more initial steps.The clarification steps, which generally comprise to use, includes following single or its various combination What one or more steps removes cell and/or cell fragment, for example, centrifuge and in-depth filtration, precipitation, flocculating And sedimentation.Clarification steps generally include to remove one or more undesirable materials, and typically expect including capture Target molecule the step of before carry out.Another crucial aspect of clarification is to remove soluble and insoluble component in sample (it can cause the pollution result of sterilizing filter in purification process later), so that whole purification process is more economical.One It is such as logical the invention provides the improvement of the conventional clarification steps for being generally used in various purification schemes in a little embodiments Cross what the turbidity/impurity and the higher throughput of downstream filter of the reduction of embodiment described here were proved.
Referred to as term used herein " chromatography " any kind of by analyte (such as target point of interest Son) with other molecules are separated present in mixture technology.Generally, analyte of interest is single point by mixture Son passes through the difference of the migration rate of mounting medium in the presence of mobile phase, or in combination and elution process, with other Molecule separation.
Term " chromatography resin " or " chromatographic media " refer to such any species in this used interchangeably Phase (such as solid phase), it separates analyte (such as target molecule) of interest with other molecules present in mixture Come.Generally, the analyte of interest is to be consolidated by the individual molecule of mixture in the presence of mobile phase through fixed The difference of the migration rate of body phase, or combining with elution process, separated with other molecules.Various types of chromatograms The example of method medium include for example cationic ion-exchange resin, affinity resin, anion exchange resin, anion-exchange membrane, dredge Aqueous interaction resin and ion exchange monolithic (monolith).
Such method is commonly referred to as term used herein " capture step ", it is used to use stimulating responsive Polymer or chromatography resin carry out combining target molecule, and it is heavy containing target molecule and the polymer or resin to which create The solid phase of starch.Typically, the target molecule is then reclaimed using elution step, and the elution step removes mesh from solid phase Molecule is marked, thus causes the separation of target molecule and one or more impurity.In various embodiments, the capture step can be with Use chromatographic media such as resin, film or single piece of material or polymer such as stimulating responsive polymer, polyelectrolyte Or the polymer of combining target molecule is carried out.
The compound for referring to being formed by the interaction of bronsted lowry acids and bases bronsted lowry as term used herein " salt ".It can be used in The various salt in various buffer solutions in methods described herein include but is not limited to acetate (such as sodium acetate), citrate (such as sodium citrate), chloride (such as sodium chloride), sulfate (such as sodium sulphate) or sylvite.
Such compound is referred to as the term " multivalent salts " being used interchangeably herein or " multivalent ion ", it is included Group more than an electric charge or containing more than an electric charge.In some embodiments, (it leads the multivalent salts as stimulation The change of solubility for having caused polymer to be caused because being stimulated in response to salt) typically result in the polymer and be precipitated out from solution.Energy The Exemplary multivalent salt enough used includes such as phosphate and sulfate.Present invention additionally comprises the counter ion counterionsl gegenions that can be used for example Such as citrate.Multivalent salts can be added to containing of interest when as stimulation described herein as independent reagent Biomolecule and stimulating responsive polymer sample in.Selectable, the salt may be coupled on substrate such as film. In a kind of specific embodiment, film is modified with polyvinyl phosphoric acid salt, it is a kind of polymer coating containing multivalent salts. Relative to other stimulation such as temperature and pH, the multivalent ion as stimulation described herein be considered as protein structure and Stability has relatively low harm.
In some embodiments, thus multivalent salts can be combined with one or more matter interactions to be formed Material or complex compound.Therefore, such salt may also be referred to as " salt for forming complex compound ".Formed complex compound salt and they The non-limiting example of the complex compound that is formed is polyvalent cation such as Cu2+And Ca2+In them and ethylenediamine tetraacetate Hydroxy-acid group complex compound;Multivalent anions such as phosphate radical (PO4 3-) and citrate and they with PAH in The complex compound of primary amine;With ion-associativity salt such as perchlorate, lauryl sulfate and dodecyl benzene sulfonate and it Complex compound with the primary amine in PAH.
" ion-associativity salt " is the salt of monovalent (cation or anion), large volume and electric charge-scattered.At some In embodiment, ion-associativity salt is used as stimulating, which results in the change of the polymer solubility to salt stimuli responsive, Thus the polymer is caused to be precipitated out from solution.The exemplary ion that can be used-associativity salt includes such as perchloric acid Salt, lauryl sulfate, dodecyl benzene sulfonate, tetraphenyl borate salts and hexanitro diphenol amine.
It is commonly referred to as dissolving as term used herein " solvent " or other scattered one or more materials To provide the liquid substance of solution.Solvent includes aqueous solvent and organic solvent, wherein useful organic solvent is including nonpolar Solvent, ethanol, methanol, isopropanol, acetonitrile, hexylene glycol, propane diols and 2,2- thiodiglycol.
As " pI " or " isoelectric point " for the term polypeptide being used interchangeably herein refer to the positive charge of polypeptide with it PH when negative electrical charge is balanced.PI can by the carbohydrate of the combination of polypeptide amino acid residue or sialic acid residues it is net Electric charge is calculated, or can be measured by equipotential focusing.
The present invention further illustrates that it is not construed as restricted by the following examples.The application is whole In cited whole bibliography, patent and Patent Application Publication content, and accompanying drawing is incorporated herein by reference.
Embodiment
Embodiment 1:Prepare unclarified non-express cell culture fluid (CCF)
In a kind of representative test, it will be given birth to from the cell of non-express Chinese hamster ovary (CHO) cell line in 10L 10x10 is grown into thing reactor (New Brunswick Scientific)6Cell/mL density, and deposited 64% Harvested during motility rate.IgG is added to concentration for 1.3g/L.The content of host cell proteins matter (HCP) uses ELISA (Cygnus# CM015) measurement is 8300ng/mL.The pH of the unclarified cell culture is pH7.2.
Embodiment 2:Synthesis includes the stimulating responsive polymer for the polyelectrolyte main chain being modified with hydrophobic group
In a kind of representative test, synthesize comprising PAH (BzMPAA) main chain being modified with hydrophobic group Stimulating responsive polymer.A kind of polymer comprising the cationic polyelectrolyte main chain being modified with hydrophobic group is to use 50% water/methanol of 10.3g linear PAHs of 40%wt (NITTOBO, 150kD), 2g lithium hydroxides and 20mL it is mixed Compound is come what is synthesized, and the mixture is stirred until being sufficiently mixed.Solution of the 2.1mL benzyl chloride in 15mL methanol is added Into the polymer solution.The mixture formed is heated 14 hours at 60 DEG C.The benzyl be modified PAH be At the end of reaction, precipitated due to incompatible with the thermodynamics of solvent.Sediment is cleaned with 30mL acetone, and it is again molten Solution is in 400mL 1M acetic acid.The polymer is precipitated by using pH7 50mM sodium phosphates to be further purified.Fig. 1 Illustrate PAH polyelectrolyte polymers and the schematic diagram of the reaction of hydrophobic group (that is, benzyl chloride).
Embodiment 3:Prepare the solution of the polyallylamine (BzMPAA) of benzyl modification
10% BzMPAA solution be by room temperature and continuously stir 16 it is small at present, the polymer of 10g embodiments 2 is molten What solution was prepared in 90g 1M acetic acid.The viscosity solution formed is slight blushing.
Embodiment 4:The polyene being modified in non-express cell culture fluid (CCF) clarification using the benzyl of various concentrations Propylamine (BzMPAA)
The 5mL that the BzMPAA of embodiment 3 is added into embodiment 1 with 0.2g, 0.3g, 0.4g and 0.5g amount is unclarified In cell culture fluid (CCF) sample.By the sample in mixed at room temperature 2 minutes.Because pH is reduced to by the addition of polymer PH4.5-5.5 scope, therefore it is 7 that the pH of the mixture is adjusted into pH using 2M Tris alkali.Add into the solution formed The two alkali formula potassium phosphates for entering 0.043g come precipitation polymers-target molecule, cell and cell fragment complex compound.By scattered solid The sediment of form of suspension is continuously mixed 5 minutes.Received by sediment and then via (4000rpm, 1 minute) is centrifuged Collection.Then supernatant from each sample is passed through 0.2um'sFilter is filtered.The purifying of gained It is listed in the table below in detail in 1.
Embodiment 5:Different pH value is used in the CCF that non-express is clarified with BzMPAA
The BzMPAA of embodiment 3 is added to four samples of the unclarified cell culture fluid of the 5mL containing embodiment 1 In (each 0.4g).By the sample in mixed at room temperature 2 minutes.Because pH is reduced to pH4.5-5.5 model by the addition of polymer Enclose, therefore it is respectively 5.5,6.5,7.5 and 8.5 that the pH of the mixture is adjusted into pH using 2M Tris alkali.It is molten to what is formed The two alkali formula potassium phosphates for adding 0.043g in liquid come precipitation polymers-target molecule, cell and cell fragment complex compound.Will be scattered Solid suspension form sediment continuously mixing 5 minutes.By sediment and then via centrifugation (4000rpm, 1 minute) It is collected.Then supernatant from each sample is passed through 0.2um'sFilter is filtered.Gained Purifying be listed in the table below in detail in 1, which depict add have non-express CHO CCF BzMPAA purifying.
Table 1.
Embodiment 6:Detect the purity level obtained in CCF clarifications using BzMPAA
The IgG for the sample for being identified to detect embodiment 4 and embodiment 5 using the HPLC of affinity protein A analysis is reclaimed Rate.The contents of IgG in the solution selectable use analytic type a-protein posts is measured.Specifically, a kind of Poros A/20 eggs White matter A posts (Applied Biosystems) are balanced with PBS, are eluted and are cleaned with 6M guanidine hydrochlorides with 0.1M lysines (pH2). IgG standard curves are produced using a series of polyclonal IgG (Seracare) of the volume injected of changes.Injected sample, and IgG concentration is determined from standard curve.
Connected using commercially available enzyme immunosorbent detection (ELISA) external member (Cygnus Technologies Inc., Southport, N.C., Cygnus#CM015) detection embodiment 4 and embodiment 5 sample host cell proteins matter (HCP). Also the sample of embodiment 4 and embodiment 5 is detected using standard pico green detections and Herring sperm DNA as reference material The DNA of product.
Embodiment 7:Prepare unclarified cell culture fluid (CCF)
The cell of expressivity Chinese hamster ovary (CHO-DG44) cell line will be derived from 10L bioreactors (New Brunswick Scientific) in grow into 10x106Cell/mL density, and harvested in 30% survival rate.It is single Clonal antibody (MAb) concentration mensuration is defined as 0.8g/L.The content of host cell proteins matter (HCP) uses ELISA (Cygnus# 3G ELISA) measurement be 200000ng/mL.The pH of the unclarified cell culture is pH6.9.
Embodiment 8:Synthesize the PAH (BzMPAA) of 20% benzyl modification
By 10g PAHs (PAA) (Nittobo, 150kD;40%wt/wt) it is placed in 100mL round-bottomed flasks, and To add 3.34g sodium hydroxides (1.2 equivalents/monomer) on a small quantity in 25mL H under room temperature and magnetic agitation2Solution in O.So Add benzyl chloride (1.38g, 1.25mL) immediately afterwards, a few minutes are stirred at room temperature, be then heated to 60 DEG C 17 hours overnight.Will be anti- Answer thing to be cooled to room temperature, remove solvent, produce polymer precipitation.The polymer with water of precipitation is cleaned, then in 1M AcOH Stirred in the aqueous solution (40mL), until realizing complete dissolving.Then by solution H2O is diluted to final volume for 400mL (1% polymer solution), stirring is lower to add two alkali formula potassium phosphate (K2HPO4) (3.48g), the pH of the solution is adjusted to about 6.8 carry out the polymer of deposition modification.The polymer is collected by being filtered in sinter funnel, and it is final in vacuum baking It is dried overnight in case at 50-60 DEG C.
Embodiment 9:Synthesize the PAH (BzMPAA) of 40% benzyl modification
By 10g PAHs (PAA) (Nittobo, 150kD;40%wt/wt) it is placed in 100mL round-bottomed flasks, and To add 3.34g sodium hydroxides (1.2 equivalents/monomer) on a small quantity in 25mL H under room temperature and magnetic agitation2Solution in O.So Benzyl chloride (2.30g, 2.09mL) is added afterwards, a few minutes are stirred at room temperature, and is then heated to 60 DEG C 17 hours overnight.By reactant Room temperature is cooled to, solvent is removed, polymer precipitation is produced.The polymer with water of precipitation is cleaned, it is then water-soluble in 1M AcOH Stirred in liquid (40mL), until realizing complete dissolving.Then by solution H2O is diluted to final volume for 400mL (1% Polymer solution), stirring is lower to add two alkali formula potassium phosphate (K2HPO4) (3.48g), the pH of the solution is adjusted to pH6.8 to sink Form sediment modified polymer.Collect the polymer by being filtered in sinter funnel, and by its it is final in vacuum drying oven 50-60 DEG C is dried overnight.
Embodiment 10:Synthesize the PAH (BzMPAA) of 60% benzyl modification
By 10g PAHs (PAA) (NITTOBO, 150kD;40%wt/wt) it is placed in 100mL round-bottomed flasks, and To add 3.34g sodium hydroxides (1.2 equivalents/monomer) on a small quantity in 25mL H under room temperature and magnetic agitation2Solution in O.So Benzyl chloride (3.23g, 2.94mL) is added afterwards, a few minutes are stirred at room temperature, and is then heated to 60 DEG C 17 hours overnight.By reactant Room temperature is cooled to, solvent is removed.The polymer with water of precipitation is cleaned, then stirred in the 1M AcOH aqueous solution (40mL), Until realizing complete dissolving.Then by solution H2O is diluted to final volume for 400mL (1% polymer solution), stirring Two alkali formula potassium phosphate (K of lower addition2HPO4) (3.48g), the pH of the solution is adjusted to the polymer that pH6.8 carrys out deposition modification.It is logical Cross and filtered in sinter funnel to collect the polymer, and it is finally dried overnight in vacuum drying oven at 50-60 DEG C.
Embodiment 11:Synthesize the PAH (DPhMPAA) that diphenyl is modified
In a kind of exemplary tests, polyelectrolyte polymers main chain PAH is modified with diphenyl.Letter and Yan Zhi, by 10g PAHs (PAA) (Nittobo, 150kD;40%wt/wt) it is placed in 100mL round-bottomed flasks, and To add 3.34g sodium hydroxides (1.2 equivalents/monomer) on a small quantity in 25mL H under room temperature and magnetic agitation2Solution in O.Then Chloro- diphenyl methane (3.68g, 3.23mL) is added, a few minutes are stirred at room temperature, 60 DEG C are then heated to 17 hours overnight.Will Reactant is subsequently cooled to room temperature, removes solvent.The polymer with water of precipitation is cleaned, then in the 1M AcOH aqueous solution Stirred in (40mL).Remaining white solid produced by hydrolysis by diphenyl methyl chloride is filtered out.Then by the solution Use H2O is diluted to final volume for 400mL (1% polymer solution), and stirring is lower to add two alkali formula potassium phosphate (K2HPO4) (3.48g), the polymer that pH6.8 carrys out deposition modification is adjusted to by the pH of the solution.Collected by being filtered in sinter funnel The polymer, and it is finally dried overnight in vacuum drying oven at 50-60 DEG C.
Embodiment 12:Synthesize the PAH (DClBzMPAA) of 6% dichloro benzyl modification
In another experiment, by 10g PAHs (PAA) (NITTOBO, 150kD;40%wt/wt) it is placed in 100mL circles In the flask of bottom, and to add 3.34g sodium hydroxides (1.2 equivalents/monomer) on a small quantity 25mL's under room temperature and magnetic agitation H2Solution in O.3,4- dichlorobenzyl chlorides (1.71g, 1.21mL) are subsequently added, it is small that mixture is stirred at room temperature overnight into 17 When.Reactant mixture is then used to 100ml H2O dilutes, and is thereafter adjusted to pH neutral (pH7.0) with phosphoric acid.By precipitation Polymer is filtered out, and uses H2O is cleaned, and is dried overnight in 60 DEG C of vacuum drying ovens.This is collected by being filtered in sinter funnel Polymer, and it is finally dried overnight in vacuum drying oven at 50-60 DEG C.
Embodiment 13:Synthesize the PAH (DClBzMPAA) of 10% dichloro benzyl modification
In another representative test, the PAH that 10% dichloro benzyl of synthesis that such as gets off is modified.By 5g polyenes third Base amine (PAA) (NITTOBO, 150kD;40%wt/wt) it is placed in 100mL round-bottomed flasks, and under room temperature and magnetic agitation To add 1.68g sodium hydroxides (1.2 equivalents/monomer) on a small quantity in 40mL 50/50H2In O/1,2 dimethoxy-ethanes (DME) Solution.3,4- dichlorobenzyl chlorides (0.57g, 0.40mL) are subsequently added, a few minutes are stirred at room temperature, 60 DEG C are then heated to overnight 21 hours.Reactant is subsequently cooled to room temperature, DME is removed in vacuum and 60-70 DEG C, remaining solvent is then removed.Will The polymer with water cleaning of precipitation, then stirs in the 1M AcOH aqueous solution (20mL), is completely dissolved until realizing.Then will Solution H2O is diluted to final volume for 200mL (1% polymer solution), and stirring is lower to add two alkali formula potassium phosphate (K2HPO4) (1.74g), the polymer that pH6.8 carrys out deposition modification is adjusted to by the pH of the solution.Collected by being filtered in sinter funnel The polymer, and it is finally dried overnight in vacuum drying oven at 50-60 DEG C.
Embodiment 14:Synthesize the PAH (DClBzMPAA) of 33% chlorobenzyl modification
In another representative test, the PAH of the chlorobenzyl modification for synthesis 33% of such as getting off.By 5g polyenes Propyl group amine (PAA) (NITTOBO, 150kD;40%wt/wt) it is placed in 100mL round-bottomed flasks, and in room temperature and magnetic agitation Under to add 3.34g sodium hydroxides (1.2 equivalents/monomer) on a small quantity in 40mL 50/50H2In O/1,2 dimethoxy-ethanes (DME) Solution.4- chlorobenzyl chlorides (1.48g) are subsequently added, a few minutes are stirred at room temperature, 60 DEG C are then heated to 21 hours overnight.The Two days, DME is removed in vacuum and 60-70 DEG C of evaporation, remaining solvent is separated with the polymer precipitated.The latter is clear with water Wash, then stirred in the 1M AcOH aqueous solution (20mL), is completely dissolved until realizing.Then by solution H2O is diluted to Final volume is 200mL (1% polymer solution), and stirring is lower to add two alkali formula potassium phosphate (K2HPO4) (1.74g), by the solution PH be adjusted to the polymer that pH7 carrys out deposition and purification.The polymer is collected by being filtered in sinter funnel, and by it most It is dried overnight eventually in vacuum and 50-60 DEG C.
Embodiment 15:Synthesize the PAH (DClBzMPAA) of 13% phenyl-benzyl modification
The PAH that the phenyl-benzyl of synthesis 13% that such as gets off in another experiment is modified.By 4.7g polyallyls Amine (PAA) (Nittobo, 150kD;40%wt/wt) be placed in 100mL round-bottomed flasks, and under room temperature and magnetic agitation with A small amount of 3.34g sodium hydroxides (1.2 equivalents/monomer) that add are in 40mL 50/50H2It is molten in O/1,2 dimethoxy-ethanes (DME) Liquid.4- phenylbenzyls chlorine (1g) is subsequently added, the mixture is heated overnight 20 hours at 55 DEG C.Then reactant is cooled to Room temperature, DME is removed in vacuum and 60-70 DEG C, remaining solvent is removed from the polymer of precipitation.The latter is clear with water Wash, be then stirred overnight in the 1M AcOH aqueous solution (40mL), is completely dissolved until realizing.Then by solution H2O is dilute Final volume is released for 200mL (1% polymer solution), stirring is lower to add two alkali formula potassium phosphate (K2HPO4) (1.74g), by this The pH of solution is adjusted to the polymer that pH7.0 carrys out deposition and purification.The polymer is collected by being filtered in sinter funnel, and It is finally dried overnight in vacuum and 50-60 DEG C.
Embodiment 16:Synthesize the PAH (DClBzMPAA) of 27% phenyl-benzyl modification
In another experiment, the PAH that 27% phenyl-benzyl of synthesis that such as gets off is modified.By 2.8g PAHs (PAA) (Nittobo, 150kD;40%wt/wt) it is placed in 100mL round-bottomed flasks, and with few under room temperature and magnetic agitation Amount adds 3.34g sodium hydroxides (1.2 equivalents/monomer) in 24mL 50/50H2It is molten in O/1,2 dimethoxy-ethanes (DME) Liquid.4- phenylbenzyls chlorine (1g) is subsequently added, the mixture is heated overnight 20 hours at 55 DEG C.Then reactant is cooled to Room temperature, DME is removed in vacuum and 60-70 DEG C, remaining solvent is removed from the polymer of precipitation.The latter is clear with water Wash, be then stirred overnight in the 1M AcOH aqueous solution (32mL), is completely dissolved until realizing.Then by solution H2O is dilute Final volume is released for 200mL (1% polymer solution), stirring is lower to add two alkali formula potassium phosphate (K2HPO4) (1.74g), by this The pH of solution is adjusted to the polymer that pH7.0 carrys out deposition and purification.The polymer is collected by being filtered in sinter funnel, and It is finally dried overnight in vacuum and 50-60 DEG C.
Embodiment 17:CCF is clarified with different polymer concentrations
In a kind of exemplary tests, the above-mentioned stimulating responsive polymers of embodiment 8-16 are have rated for the clear of CCF Clearly, it is prepared in the superincumbent embodiment 7 of description.By embodiment 8-16 polymer solution with 0.2g, 0.3g, 0.4g and 0.5g Amount be added in 5mL unclarified cell culture fluid sample.By the sample in mixed at room temperature 2 minutes.Because polymer The scope that pH is reduced to pH4.5-5.5 is added, therefore the pH of the mixture is adjusted to pH7 using 2M Tris alkali.To institute's shape Into solution in add 0.043g two alkali formula potassium phosphates come precipitation polymers-target molecule, cell and cell fragment complex compound. The sediment of scattered solid suspension form is continuously mixed 5 minutes.By sediment then via centrifuge (4000rpm, 1 minute) it is collected.Then supernatant from each sample is passed through 0.2 μmFilter was carried out Filter.The purifying of gained is listed in Table 2 below in detail.
Embodiment 18:Under different pH CCF is clarified with polymer
In another experiment, embodiment 8-16 polymer solution is added to containing the unclarified cell culture fluids of 5mL (as described in Example 7) in four samples (each 0.4g).By the sample in mixed at room temperature 2 minutes.Because polymer plus Enter the scope that pH is reduced to pH4.5-5.5, thus using 2M Tris alkali by the pH of the mixture be adjusted to pH be respectively 5.5, 6.5th, 7.5 and 8.5.The two alkali formula potassium phosphates that 0.043g is added into the solution formed carry out precipitation polymers-target molecule, thin Born of the same parents and cell fragment complex compound.The sediment of scattered solid suspension form is continuously mixed 5 minutes.By sediment and then warp It is collected by centrifuging (4000rpm, 1 minute).Then supernatant from each sample is passed through 0.2 μmFilter is filtered.The purifying of gained is listed in Table 2 below in detail.
Embodiment 19:CCF is clarified using the polymer of the multivalent ion stimuli responsive to different content
In another experiment, using different amounts of multivalent ion stimulate evaluate the polymer described in embodiment 8-15 for CCF purifying.Specifically, embodiment 8-15 polymer is added to containing 5mL unclarified cell culture fluid (such as embodiments Described in 7) four samples (each 0.4g) in.By the sample in mixed at room temperature 2 minutes.Because the addition of polymer reduces pH To pH4.5-5.5 scope, therefore it is respectively 5.5,6.5,7.5 and that the pH of the mixture is adjusted into pH using 2M Tris alkali 8.5.0.031-0.043g (the final phosphate concns of 50-70mM) two alkali formula potassium phosphates are added into the solution formed to sink Shallow lake polymer-target molecule, cell and cell fragment complex compound.The sediment of scattered solid suspension form is continuously mixed 5 minutes.It is collected by sediment and then via (4000rpm, 1 minute) is centrifuged.By the supernatant from each sample Then 0.2 μm is passed throughFilter is filtered.The purifying of gained is described in Table 2.
Embodiment 20:CCF is clarified using conventionally used flocculant
In another experiment, CCF is clarified using conventionally used flocculant chitosan, it is therefore an objective to produce correction data. Polymer solution (2wt%) is according to Riske, F. et al.;Jounral of Biotechnology, 128 (2007) 813-823 Described in method manufacture.The polymer solution is added to the CCF of embodiment 7 with the amount and the pH conditions of change that change In.Stimulation is not used to this polymer.The purifying of gained is described in table 2.
Embodiment 21:Evaluate using the purity level after stimulating responsive polymer precipitate C CF
In a kind of representative test, detect to detect embodiment 8-16 institutes using the HPLC of affinity protein A analysis The IgG rate of recovery for the polymer stated.The contents of IgG in the solution selectable use analytic type a-protein posts is measured.It is a kind of Poros A/20 a-proteins post (Applied Biosystems) is balanced with PBS, is eluted and used with 0.1M lysines (pH2) 6M guanidine hydrochlorides are cleaned.IgG standard curves are produced using a series of polyclonal IgG (Seracare) of the volume injected of changes. Injected sample, and the determination IgG concentration from standard curve.The immunosorbent connected using commercially available enzyme detects (ELISA) External member (Cygnus Technologies Inc., Southport, N.C., Cygnus#3G) detection embodiment 8-16 sample Host cell proteins matter (HCP).Also use standard pico green to detect and use Herring sperm DNA to come as reference material Detect the DNA of embodiment 8-16 sample.In order to evaluate the reduction of cell and cell fragment, centrifuged 1 minute in 4000rpm After measure turbidity.
The embodiment 8-16 of table 2. and 20 characteristic
Embodiment 22:Synthesize the polyethyleneimine (BzMPEI) that benzyl is modified
In another experiment, such as get off to have synthesized the polyethyleneimine stimulating responsive polymer of benzyl modification.Will 10g PEI (Aldrich, 750kD;50%wt/wt) be placed in 100mL round-bottomed flasks, and under room temperature and magnetic agitation with A small amount of addition~3.34g sodium hydroxides (1.2 equivalents/monomer) are in 25mL H2Solution in O.Be subsequently added benzyl chloride (2.30g, 2.09mL), a few minutes are stirred at room temperature, are then heated overnight 17 hours at 60 DEG C.Reactant is then cooled to room temperature, removed molten Agent.The polymer with water of precipitation is cleaned, then stirred in the 1M AcOH aqueous solution (40mL), is completely dissolved until realizing. Then by solution H2O is diluted to final volume for 400mL (1% polymer solution), and stirring is lower to add two alkali formula potassium phosphates (K2HPO4) (3.48g), the pH of the solution is adjusted to the polymer that pH6.8 carrys out deposition modification.By being filtered in sinter funnel To collect the polymer, and its final vacuum drying oven at 50-60 DEG C is dried overnight.
Embodiment 23:Synthesize the polyvinylamine (BzMPVA) that benzyl is modified
In a kind of representative embodiment, such as get off to have synthesized the polyvinylamine of benzyl modification.By 32g poly- (ethene Base amine) (PVA) hydrogen chloride (MW83500, Air Products and Chemicals Inc.) is weighed in glass container.Plus Enter 200mL H2The O and 50%NaOH for adding 26g, and stir until being sufficiently mixed.23.5g benzyl chlorides are added, and 70 DEG C mixing 16h.With the progress of reaction, solid white material is separated from supernatant.So that solid is settled, lost by being decanted Discard supernatant.Solid is dissolved in 350mL 3% acetic acid and stayed overnight.Add 360mL H2O, and mix 16h, Zhi Daorong Liquid is uniform.By using deionization (DI) H2O brings up to cumulative volume 3.2L cumulative volume, to be diluted to 1%w/v solution.Add Sodium phosphate to 50mM concentration, to cause the precipitation of polymer.The NaOH for adding 1M reaches pH6.8, and this provides other gather Compound is precipitated.Dry cake is filtered into, supernatant is abandoned.Solid is dried overnight at 70 DEG C.It is dissolved in 3% acetic acid, until producing Uniform solution, to produce 5%w/v solution.
Embodiment 24:When there is stimulation, compare modification the and unmodified polymer in clarification
By polyallylamine (PAA, Nittobo, 150kD;40%wt/wt) polymer (Bz-MPAA) with embodiment 10 adds Enter into the aqueous solution to produce final respectively 0.2%wt and 0.4% polymer concentration.Using potassium hydrogen phosphate as stimulation, And in the polymer solution for being added to PAA and embodiment 10 in different amounts, record the turbidity and property of formed aggregation Matter.As a result represent in table 3 below.
Table 3.
Embodiment 25:When there is stimulation, compare modification the and unmodified polymer in clarification
By unmodified polymer poly allylamine (PAA, Nittobo, 150kD;40%wt/wt), polyethyleneimine (PEI, Aldrich, 750kD;50%wt/wt), polyvinylamine (poly- (vinyl amine) (PVA) hydrogen chloride, MW 83500, Air Products and Chemicals Inc.) and embodiment 10 the polymer (Bz-MPAA) of modification, the modification of embodiment 22 Polymer (BzMPEI) and polymer-modified (BzMPVA) of embodiment 23 are added in the aqueous solution to produce final respectively 0.2 Weight % and 0.4 weight % polymer concentration.Using potassium hydrogen phosphate (150mM) as stimulation, and it is added to each 0.4% In polymer solution, the turbidity and property of formed aggregation are recorded.As a result it is shown in Table 4 below.
Table 4
Embodiment 26:The PAH (HC-t-BuMPAA) that synthesizing hexanoic acid and the tert-butyl group are modified
By 3.49g 6- bromocaproic acids be dissolved in the 40wt% linear polymers comprising 10ml poly- (allyl amine) (150kda, NITTOBO) and in the solution of 30ml sodium hydroxides (1M).The mixture is reacted into 18h at T=50 DEG C, product is regard as water-setting Glue is precipitated.The sediment is dissolved in 100mg/ml lithium hydroxide solutions, and shunk with the tert-butyl group containing 2.5ml The 10ml methanol mixing of glycerin ether.The mixture is then reacted into 18h at T=50 DEG C.It is saturating using 3.5kda molecular weight trapped substances Analysis pipe, described polymer solution is purified by using the deep dialysis (3 days) of deionization (DI) water.The polymer solution is most Final concentration is 7.2wt%.The schematic diagram of the synthetic reaction is represented in lower Fig. 2.
Embodiment 27:When being dissolved in TRIS buffer, sodium chloride changes for caproic acid and the tert-butyl group Property PAH (HC-t-BuMPAA) multivalence stimulate effect
600 μ l of embodiment 26 HC-t-BuMPAA is added to the 10ml containing 0,0.15 or 0.5M sodium chloride In 25mM sodium phosphates.The final pH of the solution is 11.6.The solution is titrated with 3M acetic acid, and recorded after each addition The turbidity of solution.As shown in figure 3, by adding the sodium chloride in addition to sodium phosphate, pH responses when observation is undergone phase transition Change.
Embodiment 28:Using polymer HC-t-BuMPAA CCF is clarified in different polymer concentrations
In a kind of representative test, such as got off using the polymer of the present invention and unclarified CCF is clarified.Will 178th, the HC-t-BuMPAA of 356 or 534 μ l embodiment 26 is added to the unclarified cell culture fluid of 5ml embodiment 1 (it contains 25mM sodium phosphate), and it is adjusted to pH8.7 using 25 μ l 3M acetic acid.After the polymer is added, use The final pH of the solution is titrated to 7.2 by 3M acetic acid, thus precipitates the polymer-cell complex compound.
The sediment of scattered solid suspension form is continuously mixed into other 5min.Then by centrifuging (4000rpm, 1min) collects the sediment, and supernatant is passed through into 0.2 μmFilter is filtered.No matter How is polymer concentration used, and this method generates the 100% Mab rate of recovery.
Embodiment 29:By monomer synthesizing polyethylene base amine (PVA) stimulating responsive polymer
Such as got off by monomer and synthesize a kind of stimulating responsive polymer, the polymer includes the repeat unit containing primary amine. The N- vinyl formamides (NVF) (SIGMA-ALDRICH, 98%) of 165g deionized waters and 22.5g are placed in into 250mL round bottoms to burn In bottle.The flask equipped has magnetic stirring apparatus and N2Measuring scale.The solution is stirred and used N2Rinse 0.5 hour, afterwards in addition 45 DEG C, and continuous flushing are heated in 0.5 hour.Initiator solution is by by the 2,2 ' of 0.288g-azo diisobutyl Amidine dihydrochloride (ABAP) (Aldrich) is added in 10mL deionized waters and dissolves to prepare.Burnt to the 250mL round bottoms Initiator solution is added under nitrogen atmosphere in bottle.By the solution under a nitrogen with strong stirring while, it is 1 small in 55 DEG C of heating When, then heat 2 hours, heated 1 hour at 75 DEG C thereafter at 65 DEG C.A kind of sticky homogeneous solution is obtained, and is cooled down To room temperature.Viscosity is measured by Brookfield viscosity DV-II+Pro Visocometer and (is set to 100RPM, 45% turns round Square, rotor #34).The viscosity of the solution formed is 278-350 centipoises (cP).The solution is transferred in 500mL flasks, and And with 330mL H2O dilutes, the lower 50%NaOH for adding 40g of stirring.The solution is heated 8 hours at 85 DEG C.
2 mole of phosphoric acid sodium are dripped into the polymer of hydrolysis by adding one, and solution when observing addition phosphate anion In white solid precipitates, to test the sensitiveness that small sample is stimulated for phosphate.Into the polymer solution of the hydrolysis 25% HCl is added dropwise, until reaching that pH is about 2.The solution strong stirring is stayed overnight, a kind of uniform yellow is obtained Solution.Add 100mL 4 moles of NaOH and 500mL isopropanols under agitation into the solution.The polymer is such as got off and divided From:100mL 2 mole of phosphoric acid sodium are added, and are cleaned by solid vacuum filter and with deionized water.The solid polymer is existed It is dried overnight in 65 DEG C of vacuum drying ovens.By partially dried polymer liquid nitrogen frozen, and fine powder is ground to form, further 65 DEG C vacuum drying oven in dry 24 hours.Finally, 40.7g dry powder has been reclaimed, and has been dissolved in 1 mole of acetic acid to final dense Spend for 5%w/w.
Fig. 4 gives the summary of polyvinylamine synthetic method.
Embodiment 30:Synthesize a series of hydrophobic modified polyvinylamine (PVA) stimulating responsive polymers
The PVA synthesized using embodiment 29, is gathered to produce the hydrophobic modified stimulating responsive of three kinds of difference as follows Compound.By the 5%PVA solution of 100mL embodiment 29 be placed in three 500mL glass jars it is each in, and labeled as tank 1,2 With 3.To three tanks it is each in add 100g 4 moles of NaOH under agitation.Then, 50g 1- third is added into each tank Alcohol stirs the solution as cosolvent, is then separately added into 0.74g, 1.47g and 2.94g benzyl chloride to tank 1,2 and 3 In.Three tanks are heated 16 hours at 60 DEG C.The polymer formed is each such as got off and separated from each reaction solution:With Reaction solution is adjusted to 500mL by deionized water, is 8 with 25%HCl adjustment pH, and add 100g 2 mole of phosphoric acid sodium.It is logical Addition phosphate anion is crossed, solid sediment is have collected via vacuum filter.The solid of each reaction is individually used into deionized water Cleaning, and be dissolved into 300mL 1 mole of acetic acid.The polymer is such as got off to be further purified:Adjust the pH of single solution For 7.4,2 mole of phosphoric acid sodium are added dropwise to precipitate the polymer, filters formed solid, the solid is washed, then with different Propyl alcohol is cleaned, and is dried 2 days in 65 DEG C of vacuum drying oven.By each dry polymer sample liquid nitrogen frozen, and grind Into fine powder.The quality for the dry polymer that tank 1,2 and 3 is reclaimed is 1.44,2.12 and 2.47g respectively.Single polymer is every It is individual to dissolve to produce 2% solution in 1 mole of acetic acid.
Embodiment 31:Come to be deprotected amine via the hydrolysis of poly- (the N- vinyl acetamides) of very HMW, next life Produce polyvinylamine (PVA) stimulating responsive polymer
In another exemplary tests, such as get off and be prepared for the stimulating responsive polymer of very HMW.It is non- Normal heavy polymer is commonly referred to as the polymer that molecular weight is equal to or more than 1000kDa.
A kind of stimulating responsive polymer comprising the repeat unit containing primary amine is such as preparation of getting off.In 2L glass jars, It is small at present by poly- (N- the vinyl acetamides)-linear homopolymers of mean molecule quantity 4060kDa 40g in strong stirring 16 (POLYSCIENCES, INC.) is dissolved into 0.8L deionized waters.The dense HCl of 140g are added into the solution, while continuously stirring 1 Hour.The tank is gently closed the lid, and the solution is heated 5 days at 99 DEG C, while the rotation of interval is described molten to mix Liquid.After heating 5 days, the solution is cooled to room temperature, cumulative volume is adjusted to 4L with deionized water.With 8 moles of NaOH in strength The solution is adjusted to pH7 under stirring.The product of hydrolysis is precipitated by the way that 2 mole of phosphoric acid sodium are added dropwise, until not seeing Untill observing other sediment.White depositions are cleaned with deionized water, and compress to remove unnecessary water.It will reclaim Polymer in 65 DEG C of vacuum drying ovens dry 2 days.By dry polymer liquid nitrogen frozen, and grind to form fine powder.Return The dry mass of receipts is 42.5g.2% solution is manufactured by the way that the dry powder is dissolved into 1 mole of acetic acid and 0.08%HCl 's.By the solution formed compared with 2% parent material (poly- (N- vinyl acetamides)-linear homopolymer) solution its for The response that phosphate or citrate are stimulated.This is arrived by the way that 2 mole of phosphoric acid sodium or 0.2 molar citric acid sodium are added dropwise Carried out in both samples of 50mL parent material and the polymer of the hydrolysis formed.
The polymer of the hydrolysis formed precipitates into whiteness when adding phosphate radical or citrate ion, and rises The solution of beginning material is not precipitated when phosphate radical or citrate ion is added dropwise, and is indicated above the parent material It is non-response for stimulating (such as multivalent anions such as phosphate radical or citrate), and as described in this embodiment The very heavy polymer of synthesis is for stimulating (such as multivalent anions such as phosphate radical or citrate) to be in response to Property.
The method that Fig. 5 illustrates a kind of deprotection of polyamine polymer, thus results in stimulating responsive polyethylene Base amine (PVA).
Embodiment 32:The polyvinylamine that poly- (N- vinyl acetamides) synthesizing hydrophobic based on deprotection is modified (PVA) stimulating responsive polymer
Poly- (N- vinyl acetamides)-linear homopolymers of 4060kDa using the deprotection by embodiment 31 and obtain PVA, such as get off the preparation very hydrophobic modified stimulating responsive polymer of HMW.
Poly- (N- vinyl acetamides) solution of the 4060kDa of 100g 2% deprotection is placed in glass jar.To the glass 100g 4 moles of NaOH adjustment pH to about 13 is added in glass tank.Then, the 1- propyl alcohol for 20g being added into tank is used as cosolvent. It is eventually adding 0.58g benzyl chloride, and by the cover upper cover.Reactant is heated 3 hours in the case where strength is rocked at 60 DEG C.3 After hour, reactant mixture is cooled to room temperature, product acetone precipitation is then collected.By the solid formed spend from The cleaning of sub- water, is then cleaned with isopropanol, and in 65 DEG C of kiln drieds 2 days.Drying solid is ground to form into fine powder, and institute The final polymer dry mass collected is 1.44g.2% solution be by by dry powder be dissolved in 1 mole of acetic acid and Manufactured in 0.08%HCl.Gathered by the way that 2 mole of phosphoric acid sodium or 0.2 molar citric acid sodium is added dropwise to the 0.5% of 5mL In polymer solution sample, to test the sensitiveness that formed solution is stimulated for multivalent ion.By add phosphate radical or Citrate ion, it was observed that white depositions, be indicated above the polymer stimulates being in response to property for multivalent anions.
Fig. 6 provides the signal of synthetic method described in this embodiment.
Embodiment 33:Synthesize stimulating responsive vinyl amine/vinyl butyl ether copolymer (VA- copolymerization-VBE)
In another experiment, a kind of stimulating responsive copolymer is prepared for, it, which has, includes the list of hydrophobic group One of body unit.
Following to synthesize stimulating responsive polymer by monomer, it includes the repeat unit containing primary amine and butyl ether.By 90g Octane, 2.5g Span-85 (SIGMA), 16g N- vinyl formamides (NVF) (ALDRICH, 98%), 5g N- butyl second Alkene ether (SIGMA) and 30g deionized waters are placed in 250mL round-bottomed flasks.The flask equipped has magnetic stirring apparatus and N2Measurement Chi.As temperature is increased to 55 DEG C, the solution is stirred and used N2Rinse 1 hour.
Initiator solution is by the way that the 2,2 ' of 0.10g-azo diisobutyl amidine dihydrochloride (ABAP) (ALDRICH) is added Enter into 10mL deionized waters, and dissolve to prepare.The initiator solution is loaded containing anti-in the case where nitrogen rinses atmosphere In the 250mL round-bottomed flasks for answering solution.By the solution in the case where strong stirring and continuous nitrogen are rinsed, heated 1 hour at 55 DEG C, Then heated at 60 DEG C 1 hour, heat 1 hour and then heated 1 hour at 80 DEG C at 70 DEG C thereafter.This generates one kind the bottom of at Portion has the two phase liquid of viscogel layer.Top layer is decanted and discarded.The addition 200mL deionized waters into bottom, and 20g 50%NaOH is added under strong stirring.The solution is heated 6 hours at 80 DEG C.After 6 hours, stop heating the solution, and And it is cooled to room temperature.Volume is increased into 2L with deionized water.By product by the way that 2 mole of phosphoric acid sodium are added dropwise, produce Big white depositions are separated.Sediment is collected by the way that supernatant is decanted, and sediment is clear with deionized water Wash.The polymer of separation is dissolved in 500mL deionized waters, 10g acetic acid and the dense HCl of 2g.By by 2 mole of phosphoric acid sodium or 0.2 molar citric acid sodium is added dropwise in 5mL 0.5% polymer solution sample, to test formed solution for multivalence The sensitiveness that ion is stimulated.White depositions are observed when adding phosphate radical or citrate ion, the polymerization is indicated above Thing stimulates being in response to property for multivalent anions.
Fig. 7 illustrates a signal of the reaction described in this embodiment.This embodiment confirm containing with hydrophobicity The copolymer of the amine of monomer copolymerization or electrically charged functionality stimulates being in response to property for multivalent ion.
Embodiment 34:Very high molecular weight polyethylene base amine is synthesized by NVF monomers via inversed emulsion polymerization method (PVA) stimulating responsive polymer
Stimulating responsive polymer is synthesized by monomer as follows, it includes the repeat unit containing primary amine.By 90g octanes, 2.5g Span-85 (SIGMA), 16g N- vinyl formamides (NVF) (ALDRICH, 98%) and 30g deionized waters are placed in In 250mL round-bottomed flasks.The flask equipped has magnetic stirring apparatus and N2Measuring scale.As temperature is increased to 55 DEG C, by the solution Stir and use N2Rinse 1 hour.Initiator solution is by by the 2,2 ' of 0.20g-azo diisobutyl amidine dihydrochloride (ABAP) (ALDRICH) is added in 20mL deionized waters and dissolves to prepare.The initiator solution is rinsed in nitrogen It is fitted under atmosphere in the 250mL round-bottomed flasks containing reaction solution.By the solution under strong stirring and continuous nitrogen purification, It is heated to 60 DEG C 2 hours, 75 DEG C is heated to thereafter 1 hour.Have the two of viscogel layer to mix in bottom this generates a kind of Liquid.Top layer is decanted and discarded.Into bottom add 500mL deionized waters, and under strong stirring add 48g 50% NaOH.The solution is heated 16 hours at 80 DEG C.After 16 hours, stop heating the solution, and be cooled to room temperature.Spend Volume is increased to 1L by ionized water.By product by the way that 2 mole of phosphoric acid sodium are added dropwise, produce big white depositions to be divided From.Sediment is collected by the way that supernatant is decanted, and sediment is cleaned with deionized water, is soaked 2 hours in isopropanol, Finally cleaned again with deionized water.The solid matter formed is dried 3 days in 65 DEG C of vacuum drying ovens.By dry polymerization Thing liquid nitrogen frozen, and fine powder is ground to form, it is further dried 1 day.The quality of the dry powder formed is 21.5g.2% solution is Manufactured by the way that the dry powder is dissolved in 1 mole of acetic acid and 0.08% HCl.By the way that 2 mole of phosphoric acid sodium or 0.2 are rubbed You are added dropwise in 50mL 1% polymer solution sample sodium citrate, to test formed solution for multivalent ion The sensitiveness of stimulation.When adding phosphate radical or citrate ion, it was observed that white depositions.
Embodiment 35:Synthesize hydrophobic modified polyvinylamine (PVA) stimulating responsive polymer of HMW
Using obtained from BASF Lupamin9095 solution (linear polyethylene base amine, average MW=340kDa, 20% solid, PH7-9), hydrophobic modified stimulating responsive polymer is produced as follows.By 300g Lupamin9095, (about 60g's is poly- Vinyl amine) it is added in 2L glass jars.Then, 40g NaOH grains and 500mL deionized waters are dissolved, and added In the tank.500mL 1,2- dimethoxy-ethanes (SIGMA) are added afterwards as cosolvent, by the solution strong stirring until Uniformly.Then, 17.66g benzyl chlorides (ACROS ORGANICS, 99%) are added in the retort under agitation.This is molten Liquid is heated to 60 DEG C 16 hours under magnetic stirring.Then the solution is cooled to room temperature, and be transferred in 5L beakers.Connect , 1500mL deionized waters are added under agitation.PH is adjusted to 5 with glacial acetic acid.By product by being slowly added to the 2 of 250mL Mole of phosphoric acid sodium is precipitated, and collects solid, is cleaned with deionized water.
The polymer is further purified by following method.The solid is dissolved in 2L 1 mole of acetic acid under agitation In.Cumulative volume is increased into 10L with deionized water, and 50% NaOH is added dropwise pH is adjusted to 7.Product is passed through 600g 2 mole of phosphoric acid sodium are added to precipitate.Solid is separated via vacuum filter, and is cleaned with deionized water.By institute The solid matter of formation is dried 3 days in 65 DEG C of vacuum drying oven.By the polymer liquid nitrogen frozen of the drying, and grind to form thin Powder, is further dried 1 day.The quality of the dry powder formed is 46g.Small sample is dissolved in 1 mole of CD3COOD/D2In O acid, Obtain1H-NMR chromatograms, it is represented in fig. 8.Will1H-NMR peaks are integrated, and it is 18% to measure benzyl modification amount.
Embodiment 36:The 200g scales synthesis of the hydrophobic modified stimulating responsive polymer based on PAH
Here in a kind of described exemplary tests, it confirms that polymer described here can be manufactured on a large scale.
Use polyallylamine solution (PAA, NITTOBO, 150kD;40%wt/wt), produce as follows hydrophobic modified Stimulating responsive polymer.By 500g polyallylamine (PAA, NITTOBO, 150kD;40%wt/wt) (about 200g's is poly- Allyl amine) it is added in 4L glass jars.Then, by 80g NaOH grains and 1000mL deionized water dissolving, and addition should In tank.1000mL 1,2- dimethoxy-ethanes (SIGMA) are added afterwards as cosolvent, by the solution strong stirring until equal It is even.Then, 114g benzyl chlorides (ACROS ORGANICS, 99%) are added in the retort.By the solution in magnetic agitation Under 60 DEG C heat 16 hours.The solution is cooled to room temperature, and is transferred in 10L beakers.
Then, 1000mL deionized waters are added under agitation, and viscous solid material is settled out from solution.The product is led to Cross and be slowly added to 200mL 2 mole of phosphoric acid sodium further to precipitate, collect solid, and cleaned with deionized water.This is polymerize Thing is further purified by following method.By the solid stirring and dissolving in 3L 1 mole of acetic acid.Will be overall with deionized water Product increases to 10L, and 50%NaOH is added dropwise pH is adjusted into 7.By the product by adding 800g 2 mole of phosphoric acid sodium To precipitate, solid is collected, and cleaned with deionized water.The polymer is even further purified by following method. By the solid stirring and dissolving in 3L 1 mole of acetic acid.Cumulative volume is increased into 10L with deionized water, and by adding dropwise Enter 50%NaOH and pH is adjusted to 7.The product is precipitated by adding 800g 2 mole of phosphoric acid sodium, solid is collected, and Cleaned with deionized water.The solid matter formed is dried 3 days in 65 DEG C of vacuum drying ovens.By the polymer liquid of the drying Chilled nitrogen, and fine powder is ground to form, it is further dried 1 day.The quality of the dry powder formed is 250g.Small sample is dissolved in 1 Mole CD3COOD/D2In O acid, obtain1H-NMR spectrum, as shown in Figure 9.Will1H-NMR peaks are integrated, and measurement benzyl modification amount is 33%.
Embodiment 37:It is determined that improving stimulating responsive polymer compared to cationic polyelectrolyte in Chinese hamster ovary celI culture In dosage when, their flocculating performance and supernatant quality
In a kind of exemplary tests described herein, the stimulating responsive polymer for comparing the present invention gathers with known Compound (that is, chitosan) it is some it is desirable that performance.
Chinese hamster ovary celI culture is prepared using the method described in embodiment 1.(MMW shells gather intermediate molecular weight chitosan Sugar) the 2%w/w solution of (Sigma-Aldrich) prepared in 1 mole of acetic acid.It is hydrophobic modified based on polyallyl The 2%w/w solution of stimulating responsive (33%-BnPAA) polymer of amine is prepared according to embodiment #36.By 10mL's Chinese hamster ovary celI culture is distributed in 15mL conical tube.For each LMW chitosan and 33%-BnPAA, contain to each Added in the conical tube for having Chinese hamster ovary celI culture single dose of polymer 0.0,0.1,0.2,0.03,0.04,0.05,0.06, 0.07th, 0.08,0.09,0.1,0.14,0.18,0.22 and 0.4%w/v.Come for the only conical tube containing 33%-BnPAA Say, pH is adjusted to 7.2, and apply the stimulation of 150mM sodium phosphates.Whole conical tubes is centrifuged 2 minutes in 3000RPM, Supernatant is decanted, and measures turbidity.
Table 5 below and Figure 10 summarize the result of representative test, to confirm that (such as shell gathers non-stimulated responsive polymer Sugar) injectivity optimizing is needed to carry out effective flocculating, and the stimulating responsive polymer of the present invention does not show to need agent Amount optimization.In other words, in the case of non-stimulated responsive polymer such as chitosan, once by the polymer of optimal dose Add effectively to carry out flocculating, then cause turbidity to raise more than optimal dose, this is undesirable.And for example described here These stimulating responsive polymers in the case of, no matter whether dosage increase, the stimulating responsive polymer has all been generated Flocculant/precipitating reagent of effect.
Table 5
Embodiment 38:It is determined that in the presence of multivalent anions stimulation, relative to non-stimulated, improving stimulating responsive polymer Flocculating performance and supernatant quality during dosage in Chinese hamster ovary celI culture
Chinese hamster ovary celI culture is prepared using the method described in embodiment 1.It is hydrophobic modified based on polyallyl The 2%w/w solution of stimulating responsive (33%-BnPAA) polymer of amine is prepared according to embodiment #36.For 33%- For BnPAA, into each conical tube containing Chinese hamster ovary celI culture parallel three parts (portion is used for phosphate, and portion is used for Citrate, portion be used for it is non-stimulated) add dosage be 0.0,0.05,0.06,0.07,0.08,0.09,0.1,0.14,0.18, 0.22 and 0.4%w/v each polymer.PH is adjusted to 7.2, and applies 150mM sodium phosphates or 150mM sodium citrates thorn Swash or do not apply stimulation.Whole conical tubes is centrifuged 2 minutes in 3000RPM, supernatant is decanted, and measure centrifugation point Chaotropic turbidity.Result of the test described in this embodiment is represented in table 6 and Figure 11.
Table 6 and Figure 11 summarize the result of representative test, to confirm stimulating responsive polymer (such as in embodiment 36 Described in) can be operated as non-stimulated response flocculant (the chitosan data being similar in embodiment 37), this is not The optimization of dose of polymer is needed using when stimulating.But, such as phosphate radical or citrate are stimulated using multivalent ion, should Stimulating responsive polymer does not need injectivity optimizing, because centrifuge separating liquid turbidity is not by the increased shadow of dose of polymer Ring.
Table 6
Embodiment 39:It is determined that when improving dosage of the unmodified stimulating responsive polymer in Chinese hamster ovary celI culture Flocculating performance and supernatant quality
In another experiment, unmodified stimulating responsive polymer described here is measured in Chinese hamster ovary celI culture In flocculating ability and supernatant quality.
Chinese hamster ovary celI culture is prepared using the method described in embodiment 1.It is unmodified based on PAH The 2%w/w solution of stimulating responsive polymer is by by polyallylamine (PAA, NITTOBO, 150kD;40%wt/wt) dissolve Prepared in 1 mole of acetic acid.10mL Chinese hamster ovary celI culture is distributed in 15mL conical tubes.Contain Chinese hamster ovary celI to each It is 0.0,0.05,0.06,0.07,0.08,0.09,0.1,0.14,0.18,0.22 and that dosage is added in the conical tube of culture 0.4%w/w each polymer.PH is adjusted to 7.2, and applies the stimulation of 150mM sodium phosphates.Whole conical tubes exist 3000RPM is centrifuged 2 minutes, supernatant is decanted, and measure turbidity.The result of the experiment of embodiments described herein is represented In table 7.
Table 7
Embodiment 40:Determine flocculating performance of the stimulating responsive polymer in Chinese hamster ovary celI culture, the sedimentation time and Supernatant quality
Chinese hamster ovary celI culture is prepared using the method described in embodiment 1.It is hydrophobic modified based on polyallyl The 10%w/w solution of stimulating responsive (33%-BnPAA) polymer of amine is similar to embodiment #36 to prepare.By 50mL's The parallel two parts of ground of Chinese hamster ovary celI culture is placed in 100mL glass cylinders.In a graduated cylinder, the stimulating responsive polymer adds Enter the dosage to 0.5%, 2 moles of Tris alkali are added dropwise pH is adjusted to 7, add 2 mole of phosphoric acid sodium solutions sodium phosphate is dense Degree is adjusted to 50mM, and solution is stirred 2 minutes.Carry out sodium phosphate and pH adjustment carry out sedimentation cell, it is cell fragment, impurity, residual The complex compound of remaining polymer and the stimulating responsive polymer;Carry out the flocculating solid;With improve granularity.Added in sodium phosphate After pH adjustment, big aggregate particle is observed in the feed that the stimulating responsive polymer is treated.By another graduated cylinder Stirring 2 minutes, is added without anything.Two graduated cylinders are stood 1 hour.At the end of 1 hour, supernatant is suctioned out, records turbid Degree.
Have been observed that the solid phase in the graduated cylinder with thorn photosensitive polymerization thing relative to the solid phase in untreated graduated cylinder (which creating indefinite Settling Front), sedimentation it is faster and (clear with clear and definite Settling Front Transformation from solid to liquid phase).
The result tested as one is summarised in table 8.Settling Front test is for untreated graduated cylinder For be a "ball-park" estimate because the Settling Front are very scattered and indefinite.
Table 8
Embodiment 41:Use the comparison of the Clarification Performance of the polyamine of different molecular weight
A series of polymer of different molecular weights is obtained, is modified, and is used to flocculate, precipitates and purifying cells training Support thing.Obtain and/or be modified by following method molecular weight for 15kD, 85kD, 150kD, 350kD, 600-950kD and The 2000-4000kD polymer with primary amine repeat unit.
15kD polyallyls amine polymer is obtained from NITTOBO, and has carried out benzyl using the method similar to embodiment 36 Base (is covalently attached benzyl and purified).Polyvinyl amine polymer Benzylation 85kD is prepared by embodiment 23 's.PAH Benzylation 150kD is prepared according to embodiment 36.Polyvinylamine polymerization Benzylation 350kD Thing is prepared by embodiment 35.950kD polyvinylamine main polymer chains are the alkali by using 2 equivalents in 80 DEG C of hydrolysis It is prepared by Polymin VZ (BASF) 8 hours.Unmodified 2000-4000kD PVA are prepared according to embodiment 31.Benzyl The 2000-4000kD polymer of base is prepared according to embodiment 32.The polymer is used for flocculating, precipitation and purifying big About 12x106Cell/mL CHO DG44 cell cultures, harvesting survival rate<50%.The flocculating is in 0.2% He What 0.4%w/w polymer metering was carried out.Being applied with the solution of 50mM sodium phosphates stimulates and is adjusted pH with 2 moles of Tris alkali To 7.
Floc size observed by record, and be expressed as+it is small flocculate, +++ ++ it is very big aggregation.It is small Bottle mark +++ ++ it is the complete region-wide separation bigger than aggregation liquid suspension.It is also noted that larger aggregation/particle sinks Drop faster, with apparent solid liquid interface.As a result it is shown in Table 9 below, which confirms the polyamine using different molecular weight Flocculating result.
Table 9.
Embodiment 42:Use the comparison of the Clarification Performance of different hydrophobic modified polyamine
Chinese hamster ovary celI culture is prepared using the method described in embodiment 1.By described in embodiment 31,32,33 and 35 Polymer samples be added in the cell culture described in embodiment 38, difference is as follows:The dose of polymer is 0.1wt%-0.6wt%, as shown in table 10.Initial cell culture turbidity is~900NTU, the centrifugation without polymer treatment The turbidity of machine separating liquid is 212NTU.PH is adjusted to 7.2, and applies the stimulation of 50mM sodium phosphates.Whole conical tubes exist 3000RPM is centrifuged 2 minutes, and supernatant is decanted, and determines centrifuge separating liquid turbidity.Experiment described in this embodiment Result be shown in Table 10 below, which confirms the different hydrophobic modified performances in different dose of polymer.
Table 10 confirms the property and/or polymer molecular weight by changing hydrophobic group, can change for stimulating Response and the turbidity of centrifuge separating liquid that is formed.
Table 10.
Embodiment 43:Influence of the reduction of the turbidity obtained with stimulating responsive polymer for downstream filter
In order to evaluate the stimulating responsive polymer for follow-up centrifugation and the influence of depth filtration step, carry out Following process.By DG44 Chinese hamster ovaries (CHO) cell line expressivity PTG1 antibody in 10L bioreactors (NEW BRUNSWICK SCIENTIFIC) in grow into about 15x106Cell/mL density, and<Harvested during 50% survival rate. Hydrophobic modified stimulating responsive (33%-BnPAA) polymer based on PAH is prepared similar to embodiment 36 10%w/w solution.
By a part for DG44CHO cell cultures with 0.2% stimulating responsive polymer 33%-BnPAA processing, and The DG44CHO cell cultures of another part are not handled.The cell culture handled to the stimulating responsive polymer In, 50mM is adjusted to by 2 mole of phosphoric acid sodium being added dropwise by phosphoric acid na concn, and by the way that 2 moles of Tris are added dropwise PH is adjusted to 7.2 by alkali.Carry out sodium phosphate and pH adjustment comes sedimentation cell, cell fragment, impurity, the complexing of residual polymer Thing and the stimulating responsive polymer, and carry out solid described in flocculating and to improve granularity.In sodium phosphate addition and pH adjustment Afterwards, big flocculate particles are observed in the feed of stimulating responsive polymer processing.Two parts are centrifuged in 3000RPM Separation 5 minutes, is decanted supernatant, determines turbidity.The centrifuge separation of the feed of measured 33%-BnPAA polymer treatments Liquid turbidity is 10NTU, and the centrifuge separating liquid turbidimetric analysis turbidimetry of untreated feed is 60NTU.
The deep bed filter throughput being each fed is determined by following method.It is 23cm by surface area2's X0HCThe disposable deep bed filters of Pod (MILLIPORE) are used to each be fed.The deep bed filter is equipped There is peristaltic pump and in line pressure sensor.By filter instruction deionized water rinsing to specifications, and will feed with 100LMH pumpings pass through, and filtrate is merged.The merging turbidity of the feed filtrate of 33%-BnPAA polymer treatments is 6NTU, not The merging turbidity of the filtrate of the feed of processing is 9NTU.The filter throughput of the feed of 33%-BnPAA polymer treatments exists It is 1304L/m during 10psi2, stop experiment due to the limitation of feed at this moment.The filter throughput of untreated feed exists It is 206L/m during 20psi2, at this moment because the limitation of pressure stops experiment.
Embodiment 44:With stimulating responsive polymer, then captured with affinity resin, to purify simulated albumin mass flow
In order to preferably evaluate influence of the stimulating responsive polymer for follow-up purification step, it is prepared for simulation and supplies Material, and carry out following process.Chinese hamster ovary celI culture is prepared using the method similar to embodiment 1.Initial feed HCP levels are~210000ppm.The hydrophobic modified stimuli responsive based on PAH is prepared similar to embodiment 36 The 10%w/w solution of property (33%-BnPAA) polymer.By a part Chinese hamster ovary celI culture with 0.1% stimulating responsive Polymer 33%-BnPAA processing, and by the Chinese hamster ovary celI culture of another part with 0.4% stimulating responsive polymer 33%-BnPAA processing, by the Chinese hamster ovary celI culture of Part III without processing.Handled to the stimulating responsive polymer In cell culture, 50mM is adjusted to by 2 mole of phosphoric acid sodium being added dropwise by phosphoric acid na concn, and by being added dropwise PH is adjusted to 7.2 by 2 moles of Tris alkali.Carry out sodium phosphate and pH adjustment comes sedimentation cell, cell fragment, impurity, remnants and gathered The complex compound of compound and the stimulating responsive polymer, and carry out solid described in flocculating and improve granularity.In sodium phosphate addition After pH adjustment, big flocculate particles are observed in the feed of stimulating responsive polymer processing.By the thin of each part Born of the same parents' culture is centrifuged 5 minutes in the centrifugal barrel of laboratory scale in 3000RPM.Record the turbid of each centrifuge separating liquid Degree, and be reported in table 11.Centrifuge separating liquid is passed through 0.2 μmFilter is filtered.
Filtrate consolidated material is purified by three steps based on chromatographic, three steps chromatography purifying is by albumen Matter A affinity chromatographies (ProSep Ultra), with reference to and elution cation-exchange chromatography (ProRes) and Flow through the membrane adsorbent anion-exchange chromatography of patternComposition.The purifying is in chromatography work station On, method in table 12 is carried out.For the consolidated material of each step, pass through elisa assay host cell proteins matter (CHOP), by the a-protein (L ProA) of elisa assay leaching, pass throughRemaining DNA is tested and analyzed, Turbidity, aggregation body protein (AGG) percentage are analyzed by size exclusion (size exclusion) HPLC, and absorbed by UV Analysing protein concentration.
Table 11.
Table 12.
A-protein
Column dimension 0.66X14cm
Method
Step Buffer solution Duration (column volume) Residence time
EQ The salt solution of phosphate-buffered 5 3
Load The feed of 0.9mg/mL clarifications 214mL 4
Cleaning The salt solution of phosphate-buffered 9 3
Elution 50mM acetic acid, pH3.0 6 3
Acid stripping (Acid Strip) 150mM phosphoric acid 3 3
EQ The salt solution of phosphate-buffered 5 3
Embodiment 45:With stimulating responsive polymer, simulated albumin matter is then purified with cationic ion-exchange resin capture Stream
In order to preferably evaluate influence of the stimulating responsive polymer for subsequent purification step, following mistake is carried out Journey.By DG44 Chinese hamster ovaries (CHO) cell line expressivity PTG1 antibody in 10L bioreactors (New Brunswick Scientific about 15x10 is grown into)6Cell/mL density, and<Harvested during 50% survival rate, HCP levels are ~142000ppm.
Hydrophobic modified stimulating responsive (the 33%- based on PAH is prepared similar to embodiment #36 BnPAA) the 10%w/w solution of polymer.By a part for Chinese hamster ovary celI culture with 0.1% stimulating responsive polymer 33%-BnPAA processing, and by the Chinese hamster ovary celI culture of another part with 0.4% stimulating responsive polymer 33%-BnPAA Processing, by the Chinese hamster ovary celI culture of Part III without processing.The cell culture handled to the stimulating responsive polymer In, 50mM is adjusted to by 2 mole of phosphoric acid sodium being added dropwise by phosphoric acid na concn, and by the way that 2 moles of Tris are added dropwise PH is adjusted to 7.2 by alkali.Carry out sodium phosphate and pH adjustment comes sedimentation cell, cell fragment, impurity, the complexing of residual polymer Thing and the stimulating responsive polymer, and carry out solid described in flocculating and improve granularity.It is adjusted in sodium phosphate addition and pH Afterwards, big aggregate particle is observed in the feed of stimulating responsive polymer processing.By the cell culture of each part Centrifuged 5 minutes in 3000RPM in the centrifugal barrel of laboratory scale.The turbidity of each centrifuge separating liquid is recorded, and It is reported in table 13.Centrifuge separating liquid is passed through 0.2 μmFilter is filtered.
Filtrate consolidated material is purified by two steps based on chromatographic, two steps chromatography purifying is by combining With elution cation-exchange chromatography (ProRes) and flow through the membrane adsorbent anion-exchange chromatography of patternComposition.The purifying is that on chromatography work station, method in table 12 is carried out.For every The consolidated material of individual step, by elisa assay host cell proteins matter (CHOP), passes through the a-protein (L of elisa assay leaching ProA), pass throughRemaining DNA is tested and analyzed, turbidity, aggregation body protein are analyzed by size exclusion HPLC (AGG) percentage, and pass through UV absorption analysis protein concentrations.
Table 13.
Embodiment 46:With the modified polyethylene film surface of phosphoric acid HEMA (PAHEMA)
In another experiment, membrane modifying is added to multivalent ion stimulation, wherein the film can be used for removing remaining stimulation Responsive polymer.
PAHEMA 16% aqueous mixture is prepared according to following formula:16g PAHEMA (Aldrich# 695890,75% PAHEMA and 25% BisHEMPA), 0.2g Irgacure 2959,93.8g water.By polyethylene film (0.65um, UPDP MILLIPORE) is soaked with methanol in advance, and is exchanged in water, is handled with the PAHEMA dispensings.Should Sample is exposed to UV light, is cleaned with first alcohol and water, and dries.Being modified the weight being added in the film by this surface is 4.4%.The infrared spectrum of the film shows strong methacrylate carbonyl absorption.With methylene blue (positively charged dyestuff) The color film generates navy blue, and cyan optical density is 1.43.
Embodiment 47:With the modified hydrophilic polyethene film surface of phosphoric acid HEMA (PAHEMA)
PAHEMA 16% aqueous mixture is prepared according to following formula:16g PAHEMA (Aldrich# 695890,75% PAHEMA and 25% BisHEMPA), 0.2g Irgacure 2959,93.8g water.By hydrophilic film (0.65um, MPLC MILLIPORE) is directly contacted with PAHEMA solution.UV exposures and cleaning are carried out as described above, and generation has The film of 7.6% increment.The infrared spectrum of the film shows strong methacrylate carbonyl absorption.With methylene blue (positively charged Lotus dyestuff) colour the film and generate navy blue, and cyan optical density is 1.45.
Embodiment 48:With the modified hydrophilic polymethacrylates of phosphoric acid HEMA (PAHEMA) Resin
In another experiment, resin modified is included to stimulate, the resin of the wherein modification can be consequently used for removing residual Remaining polymer.
Prepare with the solution constituted below:60ml allyl glycidyl ethers ether (AGE), 110g 4M NaOH, 12g Sodium sulphate.60ml ToyoPearl65C media are added into the solution, and the mixture is placed in 5 DEG C of impeller 16 hours.The medium is separated, and cleaned by conventional program.Such as get off to prepare PAHEMA grafting solutions:1.0g PAHEMA, 0.06g ammonium persulfate, 9.0g water.The ToyoPearl media that 5ml AGE is modified are added into the solution.This is mixed Compound is placed in 80C blender 16 hours.Separated and cleaned by conventional program, to produce the resin of PAHEMA modifications.When When being handled with a kind of aqueous solution of 0.01% methylene blue (it is positively charged dyestuff), this product has been coloured to dark blue Color.
Embodiment 49:Stimulating responsive polymer is hydrophilic with use phosphoric acid HEMA (PAHEMA) modification The combination on property polyethylene film surface
With following test procedure, the MPLC films (embodiment 47) being modified using PAHEMA from containing 100,10 respectively, and PAH (PAA) polymer is captured in 1ppm PAA 3 kinds of solution.Membranous disc 20mm processes diameter and protected in 15cc cells In holder.PAA solution is processed in 1.5cc/s.The film from cell retainer is washed and washed out with 100mL PBSs. The film of processing is coloured with Ponceau S.
Ponceau S are a kind of electronegative dyestuffs, and it is adsorbed onto on the surface of positively charged by force.When by PAA PAHEMA When modified film is adsorbed, the surface changes into positive charge from negative electrical charge.This conversion is described by using Ponceau S colorings Process solutions and easily observe.For being to use Macbeth density with a good measurement of Ponceau S coloring degrees The measured pinkish red optical density of meter.Table 14 is illustrated on two faces of upstream and downstream of film, different processing of films (that is, loads Have PAA) pinkish red OD value.
It can be seen that for as 1ppm, whole PAA is captured in the upstream one side of film.For 15mL's For process solutions, this corresponds to the PAA/cm of 4.8 micrograms2Film surface.
Table 14.
PAA loads (PAA of processing) Upstream (upside) magenta optical density Downstream (downside) magenta optical density
It is unprocessed 0 0
100ppm 0.77 0.77
10ppm 0.75 0.70
1ppm 0.60 0
Therefore, the result based on this embodiment, the film and resin that can release modification described herein can be used for subtracting The content of few residual polymer removes residual polymer completely.
, can be most thorough according to the teaching of bibliography (which is incorporated herein by reference) cited in application documents Understand the application.Embodiment in application documents provides the explanation to embodiment of the present invention, and should not explain For restriction on its scope.Those skilled in the art will readily recognize that present invention additionally comprises many other embodiments.Whole Open source literature and invention are incorporated herein by reference with all of which herein.Contradict or differ with the application in introduced material During cause, the application will substitute such material.Citation of any references herein is it is not an admission that such bibliography is this The prior art of invention.
Unless otherwise directed, otherwise expression composition used in application documents (including claim), cell culture, place It is to use term " about " amendment that whole numerals of the amount of manage bar part etc., which are understood to be in the whole circumstances,.Therefore, unless otherwise Opposite instruction, otherwise digital parameters are that about, and the desired performance that can be obtained according to sought by the present invention changes Become.Unless otherwise directed, otherwise it is understood to refer in the term " at least " before series of elements in this is a series of Each element.Those skilled in the art recognize or can use no more than conventional experiment to determine, sheet described herein Many equivalents of invention specific embodiment.Such equivalent purpose is included in following claim.
It will be apparent to persons skilled in the art, many changes and change can be carried out to the present invention, without Depart from its spirit and scope.Specific embodiments described herein is only what is provided by embodiment, is not represented any Limitation.Its purpose be description and embodiments are merely possible to it is exemplary, the present invention really scope and spirit be Represented by following claim.

Claims (13)

1. a kind of soluble stimulating responsive polymer, it is selected from is changed by polymer and copolymer and benzyl containing allyl amine Property polymer containing allyl amine and copolymer composition group, the wherein polymer can combine and precipitate when adding and stimulating Biomolecule of interest in sample.
2. the stimulating responsive polymer of claim 1, wherein the biomolecule of interest is therapeutical peptide.
3. the stimulating responsive polymer of claim 1, wherein the biomolecule of interest is deposited together with therapeutical peptide It is the impurity in sample.
4. the stimulating responsive polymer of claim 3, the wherein impurity be selected from host cell proteins matter, endotoxin, DNA, RNA, virus, lipid, full cell and cell fragment.
5. profit requires 1 stimulating responsive polymer, the wherein stimulation is multivalent ion.
6. the stimulating responsive polymer of claim 5, the wherein multivalent ion are phosphate radical or citrate.
7. the stimulating responsive polymer of claim 3, the wherein therapeutical peptide are antibody.
8. the stimulating responsive polymer of claim 7, the wherein antibody are monoclonal antibodies.
9. a kind of method that target molecule made in sample is separated with one or more impurity, wherein this method include following step Suddenly:
(a) sample comprising target molecule and one or more impurity is provided;
(b) sample is contacted with the soluble stimulating responsive polymer of claim 1, be consequently formed polymer and it is a kind of or The complex compound of plurality of impurities;With
(c) being added in the sample is stimulated, and the complex compound is thus settled out from solution,
Thus target molecule is made to be separated with one or more impurity.
10. the method for claim 9, wherein this method further include one or more filtration steps.
11. the method for claim 9, wherein this method further include one or more chromatographic steps.
12. the method for claim 9, the wherein stimulation are multivalent ions.
13. the method for claim 12, the wherein multivalent ion are phosphate radical or citrate.
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Families Citing this family (20)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2902799B1 (en) 2006-06-27 2012-10-26 Millipore Corp METHOD AND UNIT FOR PREPARING A SAMPLE FOR THE MICROBIOLOGICAL ANALYSIS OF A LIQUID
US8569464B2 (en) 2006-12-21 2013-10-29 Emd Millipore Corporation Purification of proteins
US8362217B2 (en) 2006-12-21 2013-01-29 Emd Millipore Corporation Purification of proteins
WO2008079302A2 (en) 2006-12-21 2008-07-03 Millipore Corporation Purification of proteins
WO2009151514A1 (en) 2008-06-11 2009-12-17 Millipore Corporation Stirred tank bioreactor
EP2370561B1 (en) 2008-12-16 2019-08-07 EMD Millipore Corporation Stirred tank reactor and method
US8992668B2 (en) * 2010-03-29 2015-03-31 Fujifilm Corporation Gas separation membrane and method for producing the same, and method for separating gas mixture, gas separation membrane module and gas separation apparatus using the same
KR101827855B1 (en) 2010-05-17 2018-02-12 이엠디 밀리포어 코포레이션 Stimulus responsive polymers for the purification of biomolecules
KR20130041843A (en) * 2010-06-08 2013-04-25 이엠디 밀리포어 코포레이션 Methods of detecting residual amounts of polymers used in the purification of biomolecules
JP2015520026A (en) * 2012-06-06 2015-07-16 イー・エム・デイー・ミリポア・コーポレイシヨン Low organic extract depth filter media processed by solvent extraction method
US9587104B2 (en) 2012-06-07 2017-03-07 The University Of Queensland Release media
EP2682168A1 (en) * 2012-07-02 2014-01-08 Millipore Corporation Purification of biological molecules
JP6336473B2 (en) * 2012-12-20 2018-06-06 メルク パテント ゲゼルシャフト ミット ベシュレンクテル ハフツングMerck Patent Gesellschaft mit beschraenkter Haftung Copolymers for protein precipitation
EP2987801B1 (en) * 2013-04-16 2019-06-12 Asahi Kasei Medical Co., Ltd. Method for the removal of aggregates from a solution containing an antibody protein
CN107075447A (en) * 2014-10-31 2017-08-18 国立大学法人东京农工大学 Cell isolation method and cell capture filter
US10450345B2 (en) * 2017-06-09 2019-10-22 General Electric Company Method of isolation of polypeptide-aptamer-polymer conjugates
WO2020004583A1 (en) * 2018-06-29 2020-01-02 三菱ケミカル株式会社 Method for isolating polypeptide, method for producing polypeptide, and apparatus for purifying polypeptide
HUP1900112A1 (en) 2019-04-04 2020-10-28 Richter Gedeon Nyrt Improvement of affinity chromatography of immunoglobulins by using pre-capture flocculation
KR102504821B1 (en) * 2021-05-24 2023-03-02 한양대학교 산학협력단 Nerve stimulator electrode comprising biocompatible solid electrolyte, method of preparing same, and nerve stimulator comprising the same
US20220412008A1 (en) * 2021-06-29 2022-12-29 Solenis Technologies, L.P. N-vinyllactam-containing polymers for papermaking

Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20040127639A1 (en) * 2001-03-30 2004-07-01 Xiaoru Wang Composite colorant particles
US20060281847A1 (en) * 2005-06-10 2006-12-14 Canon Finetech Inc. Fine particle liquid dispersion and recording medium using the same
CN1938348A (en) * 2004-03-31 2007-03-28 巴斯福股份公司 Polymers comprising diallylamines
WO2008079302A2 (en) * 2006-12-21 2008-07-03 Millipore Corporation Purification of proteins
CN101420991A (en) * 2003-11-20 2009-04-29 血管技术国际股份公司 Polymer compositions and methods for their use
CN101437901A (en) * 2006-03-03 2009-05-20 罗地亚管理公司 Modification of solid surfaces by means of polymer combinations
CN101678318A (en) * 2007-05-25 2010-03-24 默克专利股份公司 Graft copolymer for cation-exchange chromatography
CN111356434A (en) * 2017-12-12 2020-06-30 莱雅公司 Composition comprising an oil and a polyionic complex comprising a cellulose-based cationic polymer having at least one fatty chain

Family Cites Families (283)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US2923669A (en) 1954-11-22 1960-02-02 Millipore Filter Corp Method of bacterial analysis
US3211645A (en) 1962-07-17 1965-10-12 Technicon Instr Method and apparatus for filtering sanguineous liquid streams
US3565973A (en) 1967-11-14 1971-02-23 Amicon Corp Purifying cross-linked polyelectrolytes
US3556302A (en) 1969-01-06 1971-01-19 Amicon Corp Filtration apparatus having flow distributor
US3632507A (en) 1970-06-30 1972-01-04 Standard Brands Chem Ind Inc Flocculation of particles dispersed in aqueous media and flocculants used therein
US3702806A (en) 1970-09-03 1972-11-14 William Emil Oliva Disposable culture media container
US3737377A (en) 1971-01-27 1973-06-05 Miles Lab Purification of lactase
GB1354349A (en) 1971-10-12 1974-06-05 Allied Colloids Mfg Flocculating agents
US3968037A (en) 1972-09-01 1976-07-06 Calgon Corporation Emulsion polymerization of cationic monomers
US4045377A (en) 1975-10-20 1977-08-30 Hercules Incorporated Cationic polymer prepared from dicyandiamide, a polyamide, a dialkylamine, and an epoxide
US4055469A (en) 1976-12-10 1977-10-25 Eastman Kodak Company Purification of microbial enzyme extracts using synthetic polyelectrolytes
FR2413974A1 (en) 1978-01-06 1979-08-03 David Bernard DRYER FOR SCREEN-PRINTED SHEETS
US4215198A (en) 1978-09-15 1980-07-29 Gordon Maurice R Sterility testing unit
US4380590A (en) 1978-09-19 1983-04-19 Rohm And Haas Company Emulsion copolymer cation exchange resins
US4359537A (en) 1978-09-19 1982-11-16 Rohm And Haas Company Emulsion copolymer anion exchange resins
US4200695A (en) 1978-09-19 1980-04-29 Rohm And Haas Company Flocs for filtration and deionization prepared from cationic and anionic emulsion ion exchange resins
US4515893A (en) 1979-04-26 1985-05-07 Ortho Pharmaceutical Corporation Hybrid cell line for producing complement-fixing monoclonal antibody to human T cells
US4305829A (en) 1979-06-29 1981-12-15 Union Carbide Corporation Process for flocculating an aqueous suspension of particles with quaternary ammonium graft copolymers
DE2934854A1 (en) 1979-08-29 1981-09-10 Basf Ag, 6700 Ludwigshafen METHOD FOR PRODUCING NITROGEN-CONDENSING PRODUCTS AND THE USE THEREOF
EP0053016A3 (en) 1980-11-20 1982-06-30 Unilever Plc Fat refining
US4317726A (en) 1981-02-12 1982-03-02 The United States Of America As Represented By The Secretary Of The Army Microbial filter assembly
DE3145320A1 (en) 1981-11-14 1983-05-26 Biotest-Serum-Institut Gmbh, 6000 Frankfurt MICROFILTRATION DEVICE FOR FILTRATION OF COAGELS AND MICROAGGREGATES OF BLOOD
CA1180827A (en) 1982-03-23 1985-01-08 Michael Heskins Polymeric flocculants
US4382028A (en) 1982-07-19 1983-05-03 Monsanto Company Separation of plasma proteins from cell culture systems
US4816567A (en) 1983-04-08 1989-03-28 Genentech, Inc. Recombinant immunoglobin preparations
US4528933A (en) 1983-05-11 1985-07-16 Robert Allen Container with indicating closure
US4828701A (en) 1983-08-25 1989-05-09 Regents Of The University Of Minnesota Temperature-sensitive method of size-selective extraction from solution
US4533496A (en) 1984-05-08 1985-08-06 Monsanto Company Method of isolating monoclonal antibodies from hybridoma cultures
US5672347A (en) 1984-07-05 1997-09-30 Genentech, Inc. Tumor necrosis factor antagonists and their use
US4780409A (en) 1985-05-02 1988-10-25 Genetic Systems Corporation Thermally induced phase separation immunoassay
US4904385A (en) 1985-05-23 1990-02-27 The Dow Chemical Company Porous filter media and membrane support means
US4676980A (en) 1985-09-23 1987-06-30 The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services Target specific cross-linked heteroantibodies
US4863613A (en) 1985-10-25 1989-09-05 Regents Of The University Of Minnesota Soy protein isolation process using swellable poly(N-isopropylacrylamide) gels
US5091178A (en) 1986-02-21 1992-02-25 Oncogen Tumor therapy with biologically active anti-tumor antibodies
US4925785A (en) 1986-03-07 1990-05-15 Biotechnica Diagnostics, Inc. Nucleic acid hybridization assays
US4912032A (en) 1986-04-17 1990-03-27 Genetec Systems Corporation Methods for selectively reacting ligands immobilized within a temperature-sensitive polymer gel
IL85035A0 (en) 1987-01-08 1988-06-30 Int Genetic Eng Polynucleotide molecule,a chimeric antibody with specificity for human b cell surface antigen,a process for the preparation and methods utilizing the same
US4839046A (en) 1987-08-20 1989-06-13 The United States Of America As Represented By The Administrator Of The National Aeronautics And Space Administration Bio-reactor chamber
AU2303588A (en) 1987-08-27 1989-03-31 Polyfiltronics Limited Filter units for biological sample preparation
US5091313A (en) 1988-08-05 1992-02-25 Tanox Biosystems, Inc. Antigenic epitopes of IgE present on B cell but not basophil surface
US5720937A (en) 1988-01-12 1998-02-24 Genentech, Inc. In vivo tumor detection assay
US5429952A (en) 1988-02-02 1995-07-04 Biocode, Inc. Marking of products to establish identity and source
EP0420937B1 (en) 1988-06-21 1994-11-09 Genentech, Inc. Therapeutic compositions for the treatment of myocardial infarction
JP2577782B2 (en) 1988-07-18 1997-02-05 富士通株式会社 Knowledge information system
US5152903A (en) 1988-12-19 1992-10-06 American Cyanamid Company Cross-linked cationic polymeric microparticles
US5340865A (en) 1988-12-19 1994-08-23 Cytec Technology Corp. Cross-linked cationic polyermic microparticles
MX18620A (en) 1988-12-19 1993-10-01 American Cyanamid Co HIGH PERFORMANCE POLYMERIC FLOCULANT, PROCESS FOR ITS PREPARATION, METHOD FOR THE RELEASE OF WATER FROM A DISPERSION OF SUSPENDED SOLIDS AND FLOCULATION METHOD OF A DISPERSION OF SUSPENDED SOLIDS
US6191242B1 (en) 1988-12-19 2001-02-20 Cytec Technology Corp. Process for making high performance anionic polymeric flocculating agents
US5354481A (en) 1988-12-19 1994-10-11 Cytec Technology Corp. Water-soluble highly branched polymeric microparticles
US4968435A (en) 1988-12-19 1990-11-06 American Cyanamid Company Cross-linked cationic polymeric microparticles
US5530101A (en) 1988-12-28 1996-06-25 Protein Design Labs, Inc. Humanized immunoglobulins
US5003047A (en) 1989-01-10 1991-03-26 Massachusetts Institute Of Technology Method for purifying biologically active ligate
US5139031A (en) 1989-09-18 1992-08-18 La Mina Ltd. Method and device for cytology and microbiological testing
WO1991000360A1 (en) 1989-06-29 1991-01-10 Medarex, Inc. Bispecific reagents for aids therapy
US5047511A (en) 1989-08-28 1991-09-10 Pitman-Moore, Inc. Method for recovering recombinant proteins
JPH03296657A (en) 1990-04-13 1991-12-27 W R Grace & Co Supporter for electrophoresis and electrophoretic method using said supporter
JPH046463A (en) 1990-04-16 1992-01-10 W R Grace & Co Carrier for liquid chromatography and liquid chromatographic method using said carrier
CA2086417C (en) 1990-06-29 1999-07-06 Biosource Technologies, Inc. Melanin production by transformed organisms
US5116754A (en) 1990-10-04 1992-05-26 Fraser Ann D E Separation of bacteria from organic matter
DE4103969A1 (en) 1991-02-09 1992-08-13 Basf Ag METHOD FOR PRODUCING FINE-PARTICLE, WATER-SOLUBLE OR WATER-SWELLABLE POLYMERISATS
US5238545A (en) 1991-02-27 1993-08-24 W. R. Grace & Co.-Conn. Electrophoretic gel for separation and recovery of substances and its use
US5171450A (en) 1991-03-20 1992-12-15 Nalco Chemical Company Monitoring and dosage control of tagged polymers in cooling water systems
JPH06507398A (en) 1991-05-14 1994-08-25 リプリジェン コーポレーション Heterogeneous conjugate antibody for treatment of HIV infection
FR2677664A1 (en) 1991-06-13 1992-12-18 Millipore Sa Device and process for the microbiological control of liquids under pressure
ES2296839T3 (en) 1991-08-14 2008-05-01 Genentech Inc. IMMUNOGLOBULIN VARIANTS FOR SPECIFIC FC-EPSILON RECEIVERS.
WO1993004713A1 (en) 1991-09-11 1993-03-18 The University Of Melbourne Method for intravenous drug infusion
ATE148889T1 (en) 1991-09-18 1997-02-15 Affymax Tech Nv METHOD FOR SYNTHESIS OF VARIOUS COLLECTIONS OF OLIGOMERS
WO1993008829A1 (en) 1991-11-04 1993-05-13 The Regents Of The University Of California Compositions that mediate killing of hiv-infected cells
US5525519A (en) 1992-01-07 1996-06-11 Middlesex Sciences, Inc. Method for isolating biomolecules from a biological sample with linear polymers
EP1514934B1 (en) 1992-02-06 2008-12-31 Novartis Vaccines and Diagnostics, Inc. Biosynthetic binding protein for cancer marker
FR2687476B1 (en) 1992-02-13 1994-05-20 Millipore Sa DEVICE FOR VERIFYING THE STERILITY OF A FLUID.
US5258122A (en) 1992-04-16 1993-11-02 Amicon, Inc. Cross-flow filter device with pressure-balancing feature
ES2110543T3 (en) 1992-07-22 1998-02-16 Hoechst Ag DERIVATIVES OF POLI (VINYL-AMINES) THAT HAVE HYDROPHILIC CENTERS, PROCEDURE FOR THEIR PREPARATION AS WELL AS THE USE OF THE COMPOUNDS AS MEDICINES, VEHICLES OF ACTIVE SUBSTANCES AND AUXILIARY FOOD INGREDIENTS.
EP0582536A1 (en) 1992-07-27 1994-02-09 International Business Machines Corporation Method of and apparatus for providing automatic response actions for preexisting user input procedures
JP3253730B2 (en) * 1992-08-21 2002-02-04 科学技術振興事業団 Temperature-responsive physiologically active substance-oligomer complex and method for producing the same
JPH08500826A (en) 1992-08-21 1996-01-30 ジェネンテク,インコーポレイテッド Methods of treating LFA-1 mediated diseases
US5736137A (en) 1992-11-13 1998-04-07 Idec Pharmaceuticals Corporation Therapeutic application of chimeric and radiolabeled antibodies to human B lymphocyte restricted differentiation antigen for treatment of B cell lymphoma
US5324787A (en) 1992-11-18 1994-06-28 Air Products And Chemicals, Inc. Modification of poly (vinylamine)
SE9300090D0 (en) 1993-01-14 1993-01-14 Bo Gustav Mattiasson AFFINITY CLEANING WITH COMPLEX BOND LIGAND
US5374971A (en) 1993-03-12 1994-12-20 Picturetel Corporation Two-view video camera stand and support method
US5342581A (en) 1993-04-19 1994-08-30 Sanadi Ashok R Apparatus for preventing cross-contamination of multi-well test plates
DK83093D0 (en) 1993-07-09 1993-07-09 Novo Nordisk As COURSE OF ACTION
US5840851A (en) 1993-07-23 1998-11-24 Plomer; J. Jeffrey Purification of hemoglobin
US5354801A (en) 1993-08-12 1994-10-11 Cytec Technology Corp. Process for producing small polymer phase droplet microemulsions by multistep aqueous phase addition
WO1995016203A2 (en) 1993-12-10 1995-06-15 Genentech, Inc. Methods for diagnosis of allergy and screening of anti-allergy therapeutics
DE69501817T2 (en) 1994-01-18 1998-09-10 Genentech Inc METHOD FOR TREATING PARASITAL INFECTIONS USING IGE ANTAGONISTS
US5622857A (en) 1995-08-08 1997-04-22 Genespan Corporation High performance cell culture bioreactor and method
DE4406624A1 (en) 1994-03-01 1995-09-07 Roehm Gmbh Crosslinked water-soluble polymer dispersions
US5707622A (en) 1994-03-03 1998-01-13 Genentech, Inc. Methods for treating ulcerative colitis
US5512480A (en) 1994-03-11 1996-04-30 Baxter International Inc. Flow-through bioreactor with grooves for cell retention
TW474813B (en) 1994-06-10 2002-02-01 Geltex Pharma Inc Alkylated composition for removing bile salts from a patient
AU3100995A (en) 1994-07-18 1996-02-16 Gel Sciences, Inc. Responsive polymer gel beads
DK0699687T3 (en) 1994-08-31 2004-04-26 Mitsubishi Pharma Corp Process for purifying recombinant human serum albumin
GB9422504D0 (en) 1994-11-08 1995-01-04 Robertson Patricia M B Blood testing
AUPN060095A0 (en) 1995-01-13 1995-02-09 Enviro Research Pty Ltd Apparatus for biomass production
GB9503109D0 (en) 1995-02-17 1995-04-05 Hampshire Advisory Tech Serv Diagnostic test tube and kits
US5731168A (en) 1995-03-01 1998-03-24 Genentech, Inc. Method for making heteromultimeric polypeptides
IL117645A (en) 1995-03-30 2005-08-31 Genentech Inc Vascular endothelial cell growth factor antagonists for use as medicaments in the treatment of age-related macular degeneration
US5573675A (en) 1995-05-11 1996-11-12 Nalco Chemical Company Clarification of deinking process waters using polymers containing vinylamine
GB9510634D0 (en) 1995-05-25 1995-07-19 Sev Trent Water Ltd Filtration and culture methods and apparatus
US5733507A (en) 1995-06-07 1998-03-31 Inphocyte, Inc. Biological cell sample holder for use in infrared and/or Raman spectroscopy analysis holder
CA2222231A1 (en) 1995-06-07 1996-12-19 Imclone Systems Incorporated Antibody and antibody fragments for inhibiting the growth of tumors
JPH0912598A (en) * 1995-06-29 1997-01-14 Sekisui Chem Co Ltd Separation and purification of hemoglobin
US6267958B1 (en) 1995-07-27 2001-07-31 Genentech, Inc. Protein formulation
CA2230557A1 (en) * 1995-09-01 1997-03-13 University Of Washington Interactive molecular conjugates
GB2305936B (en) 1995-10-06 1997-09-03 Jonathan William Lewis Sterile, disposable culture vessels for the rapid growth of bacteria
JPH09124697A (en) 1995-11-01 1997-05-13 Toagosei Co Ltd Peptide and monoclonal antibody
US5879564A (en) 1995-11-14 1999-03-09 Cytec Technology Corp. High performance polymer flocculating agents
US5807489A (en) 1995-11-14 1998-09-15 Cytec Technology Corp. High performance polymer flocculating agents
ZA97248B (en) 1996-01-18 1997-07-18 Rohm & Haas Method for identifying and quantifying polymers utilizing immunoassay techniques
CA2242414C (en) 1996-01-23 2012-01-03 Genentech, Inc. Anti-cd18 antibodies for use against stroke
AU1989197A (en) 1996-03-06 1997-09-22 Akzo Nobel N.V. Automated nucleic acid extraction from samples
WO1997034909A1 (en) 1996-03-20 1997-09-25 Bio Merieux Nucleic acid isolation
FR2749082B1 (en) 1996-05-24 1998-06-26 Bio Merieux SUPERPARAMAGNETIC PARTICLES AND MONODISPERSEES
US7147851B1 (en) 1996-08-15 2006-12-12 Millennium Pharmaceuticals, Inc. Humanized immunoglobulin reactive with α4β7 integrin
US5994560A (en) 1996-08-29 1999-11-30 Hoechst Celanese Corp. Resolution of racemic mixtures using polymers containing chiral units
PT941344E (en) 1996-11-27 2004-10-29 Genentech Inc HUMANIZED ANTI-CD11A ANTIBODIES
SI0950067T1 (en) 1996-11-27 2007-12-31 Genentech Inc Affinity purification of polypeptide on protein a matrix
JP3722842B2 (en) 1997-01-24 2005-11-30 株式会社セルシード Method for separating PTH-amino acids
US5942444A (en) 1997-01-27 1999-08-24 Biocode, Inc. Marking of products to establish identity, source and fate
EP1012327B1 (en) 1997-01-29 2006-01-11 Pall Corporation Filtration assembly
US5929214A (en) 1997-02-28 1999-07-27 Cornell Research Foundation, Inc. Thermally responsive polymer monoliths
AU743758B2 (en) 1997-04-07 2002-02-07 Genentech Inc. Anti-VEGF antibodies
EP0981618B2 (en) 1997-05-15 2011-08-24 Genentech, Inc. Anti-apo-2 antibody
CA2289665C (en) 1997-06-13 2005-08-09 Genentech, Inc. Protein recovery by chromatography followed by filtration upon a charged layer
US5994511A (en) 1997-07-02 1999-11-30 Genentech, Inc. Anti-IgE antibodies and methods of improving polypeptides
US6007803A (en) 1997-09-19 1999-12-28 Geltex Pharmaceuticals, Inc. Ionic polymers as toxin binding agents
US6300142B1 (en) 1997-11-28 2001-10-09 Provalis Diagnostics Ltd Device and apparatus for conducting an assay
US6914137B2 (en) 1997-12-06 2005-07-05 Dna Research Innovations Limited Isolation of nucleic acids
JP4217804B2 (en) * 1998-09-14 2009-02-04 独立行政法人産業技術総合研究所 Thermally responsive separation material and drug release capsule using thermoresponsive polymer derivative having upper limit solution critical temperature
DE69839323T2 (en) 1997-12-09 2009-04-09 National Institute Of Advanced Industrial Science And Technology Stimulus-responsive polymer via keto-enol tautomerism
FR2773416B1 (en) 1998-01-06 2000-02-11 Bio Merieux IMPROVED MAGNETIC PARTICLES, PROCESSES FOR OBTAINING SAME AND THEIR USES IN THE SEPARATION OF MOLECULES
ES2319349T3 (en) 1998-05-22 2009-05-06 Cellseed Inc. CHROMATOGRAPHIC FILLING THAT HAS A CHARACTERISTIC METHOD TO SEPARATE A SUBSTANCE AND USE OF IT.
US6756217B1 (en) 1998-05-29 2004-06-29 Southern Illinois University Glass composite materials containing alkoxosilane derivative having alterable charge, hydrophobic and hydrophilic groups
DE69920711D1 (en) 1998-07-21 2004-11-04 Monsanto Technology Llc Clarification of Protein Precipitation Suspensions by Using Anionic Polymeric Flocculants
US6221655B1 (en) 1998-08-01 2001-04-24 Cytosignal Spin filter assembly for isolation and analysis
SE9802882D0 (en) 1998-08-28 1998-08-28 Amersham Pharm Biotech Ab Composite material and its use
WO2000011953A1 (en) 1998-09-01 2000-03-09 Penn State Research Foundation Method and apparatus for aseptic growth or processing of biomass
WO2000023087A1 (en) 1998-10-21 2000-04-27 Sunol Molecular Corporation Polyspecific binding molecules and uses thereof
US6641735B1 (en) 2000-03-23 2003-11-04 Japan Chemical Innovation Institute Separatory material with the use of stimulus-responsive polymer and separation method by using the separatory material
US6454950B1 (en) 1998-12-30 2002-09-24 Amersham Pharmacia Biotech Ab Separation method utilizing liquid-liquid partition
FR2788008B1 (en) 1998-12-30 2001-03-23 Inst Curie THERMOSENSITIVE MEDIUM FOR THE ELECTROKINETIC SEPARATION OF SPECIES WITHIN A SEPARATION CHANNEL
WO2000040974A1 (en) 1999-01-07 2000-07-13 Quidel Corporation Pyridinium crosslinks assay
ATE301679T1 (en) 1999-01-29 2005-08-15 Amersham Biosciences Kk TEMPERATURE-SENSITIVE POLYMER AND METHOD FOR THE PRODUCTION THEREOF
SE9900378D0 (en) 1999-02-05 1999-02-05 Forskarpatent I Syd Ab Gels with shape memory
US6565872B2 (en) 1999-02-16 2003-05-20 Xiao Yu Wu Polymeric system for drug delivery and solute separation
US6214221B1 (en) 1999-02-22 2001-04-10 Henry B. Kopf Method and apparatus for purification of biological substances
WO2000050888A1 (en) 1999-02-25 2000-08-31 Pall Corporation Chromatography devices, porous medium modules used in chromatography devices, and methods for making porous medium modules
AU3867400A (en) 1999-03-19 2000-10-09 Duke University Methods of using bioelastomers
EP1194228A1 (en) 1999-05-11 2002-04-10 Japan Chemical Innovation Institute Affinity-controlling material with the use of stimulus-responsive polymer and separation/purification method with the use of the material
US6946129B1 (en) 1999-06-08 2005-09-20 Seattle Genetics, Inc. Recombinant anti-CD40 antibody and uses thereof
US6211140B1 (en) 1999-07-26 2001-04-03 The Procter & Gamble Company Cationic charge boosting systems
EP1201667B1 (en) 1999-07-29 2005-09-21 National Institute of Advanced Industrial Science and Technology Polymerizable biotin derivatives, biotin polymer, and polymer responsive to avidin stimulation
GB9919187D0 (en) 1999-08-14 1999-10-20 Ciba Spec Chem Water Treat Ltd Flocculation of cell material
US6420487B1 (en) 1999-09-08 2002-07-16 Council Of Scientific And Industrial Research Process for the preparation of thermoprecipitating affinity polymers
US6294622B1 (en) 1999-09-27 2001-09-25 Ecole Polytechnique Federale De Lausanne (Epfl) Polymer flocculants with improved dewatering characteristics
US6258275B1 (en) 1999-10-01 2001-07-10 Ecole Polytechnique Federale De Lausanne Affinity macroligands
NZ518477A (en) 1999-10-29 2004-10-29 Genentech Inc Isolated anti-prostate stem cell antigen (PSCA) antibodies that internalise upon binding to PSCA on a mammalian cell, including a PSCA-expressing tumour cell, in vivo
US6544424B1 (en) 1999-12-03 2003-04-08 Refined Technology Company Fluid filtration system
FR2804117B1 (en) 2000-01-21 2004-08-20 Bio Merieux PROCESS FOR ISOLATING PROTEINS AND / OR NUCLEIC ACIDS, PARTICLE COMPLEXES AND PROTEINS AND / OR NUCLEIC ACIDS, REAGENT AND APPLICATIONS
AU2001246843A1 (en) 2000-04-05 2001-10-15 Japan Chemical Innovation Institute Novel material for use in separation and separating method using the same
US6372145B1 (en) 2000-06-05 2002-04-16 Power Engineering Company Fat and protein removal from process streams
JP4797144B2 (en) 2000-08-21 2011-10-19 独立行政法人産業技術総合研究所 Magnetic fine particles with lower critical solution temperature
EP1312627B1 (en) 2000-08-21 2009-10-07 National Institute of Advanced Industrial Science and Technology Polymers
EP1316599B1 (en) 2000-08-21 2014-03-26 National Institute of Advanced Industrial Science and Technology Magnetic particles and process for producing the same
EP1312643B1 (en) 2000-08-23 2005-05-11 National Institute of Advanced Industrial Science and Technology Temperature-responsive polymer/polymer complex
US6374684B1 (en) 2000-08-25 2002-04-23 Cepheid Fluid control and processing system
CA2421515C (en) 2000-09-06 2010-07-20 University Of Massachusetts High efficiency protein extraction
JP5109003B2 (en) 2000-10-13 2012-12-26 岡野 光夫 Separation material such as stimulus-responsive affinity chromatography material and separation purification method
US6605714B2 (en) 2000-11-29 2003-08-12 Council Of Scientific And Industrial Research Thermoprecipitating polymer containing enzyme specific ligands, process for the preparation thereof, and use thereof for the separation of enzymes
US6367749B2 (en) 2001-03-21 2002-04-09 Southern Imperial, Inc. Stand base
JP2003153684A (en) 2001-11-20 2003-05-27 Advance Co Ltd Three-dimensional cell culture system
GB0108548D0 (en) 2001-04-05 2001-05-23 Ciba Spec Chem Water Treat Ltd Process for flocculating suspensions
US6688487B2 (en) 2001-04-13 2004-02-10 The Coca-Cola Company Locking cup and lid with negative draft sealing surfaces
WO2002083743A1 (en) 2001-04-16 2002-10-24 Wsp Chemicals & Technology Llc Water-soluble polymer complexes
US7157603B2 (en) 2001-05-14 2007-01-02 Polytag Technology Sa Method for the separation of oligomeric N-substituted (meth)acrylamide compounds and conjugates thereof which are reversibly thermally precipitating
JP3907508B2 (en) 2001-07-30 2007-04-18 松下エコシステムズ株式会社 Microorganism collection chip, microorganism collection kit, microorganism measurement method, and microorganism measurement apparatus
JP4118927B2 (en) 2001-07-30 2008-07-16 松下エコシステムズ株式会社 Specimens for normality confirmation test of microorganism weighing device
FR2829500B1 (en) 2001-09-13 2003-12-12 Hemosystem PROCESS FOR THE CONCENTRATION AND DETECTION OF PATHOGENIC SPROUTS FROM BLOOD PRODUCTS AND / OR DERIVATIVES THEREOF AND DEVICE FOR CARRYING OUT SAID METHOD
GB0126923D0 (en) 2001-11-09 2002-01-02 Procter & Gamble Chitosan compositions
TWI288758B (en) 2001-12-19 2007-10-21 Ind Tech Res Inst Thermal responsive, water-soluble polymers
US6723245B1 (en) 2002-01-04 2004-04-20 Nalco Company Method of using water soluble cationic polymers in membrane biological reactors
CN1617938A (en) 2002-01-16 2005-05-18 戴诺生物技术有限公司 Method for isolating nucleic acids and protein from a single sample
US6998456B1 (en) 2002-02-15 2006-02-14 Iowa State University Research Foundation pH-sensitive methacrylic copolymers and the production thereof
US20040029143A1 (en) 2002-02-28 2004-02-12 Jeffrey Van Ness Cationic polyelectrolytes in biomolecule purification and analysis
US7011930B2 (en) 2002-03-15 2006-03-14 The Penn State Research Foundation Method for control of temperature-sensitivity of polymers in solution
WO2003090842A1 (en) 2002-04-26 2003-11-06 Gl Tool And Manufacturing Co.Inc. A valve
DE10224352A1 (en) 2002-06-01 2003-12-11 Mueller Schulte Detlef Thermosensitive polymer carrier with changeable physical structure for biochemical analysis, diagnostics and therapy
WO2003104383A1 (en) 2002-06-07 2003-12-18 Millipore Corporation Kit and process for microbiological for on-site examination of a liquid sample
CN100393812C (en) 2002-06-21 2008-06-11 海茂株式会社 Water-soluble polymer dispersion, process for producing the same and method of use therefor
AU2003259289A1 (en) 2002-07-30 2004-02-16 University Of Washington Apparatus and methods for binding molecules and cells
US7422724B1 (en) 2002-08-07 2008-09-09 Sandia Corporation Biological preconcentrator
SE0202552D0 (en) 2002-08-27 2002-08-27 Amersham Biosciences Ab Recovery of plasmids in an aqueous two-phase system
CN1300297C (en) 2002-09-20 2007-02-14 华东理工大学 Digestive reactor for animal cell extension inoculation
US6837610B2 (en) 2002-09-27 2005-01-04 Ilc Dover Lpp Bioprocess container, bioprocess container mixing device and method of use thereof
US6673598B1 (en) 2002-10-29 2004-01-06 Synthecon, Inc. Disposable culture bag
EP1944320A1 (en) 2002-12-16 2008-07-16 Genentech, Inc. Immunoglobulin variants and uses thereof
EP2017617A1 (en) 2002-12-23 2009-01-21 William Marsh Rice Univeristy Wound dressing
US7083948B1 (en) 2002-12-24 2006-08-01 Immunex Corporation Polypeptide purification reagents and methods for their use
TWI257928B (en) 2002-12-27 2006-07-11 Ind Tech Res Inst Method for the separation of polysaccharides
AU2004230670B2 (en) 2003-01-09 2010-11-25 Genentech, Inc. Purification of polypeptides
AU2004210964A1 (en) 2003-02-11 2004-08-26 University Of Washington Stimuli-responsive polymer conjugates and related methods
WO2004078968A1 (en) 2003-03-04 2004-09-16 Hideko Kanazawa System for evaluating drug metabolism capacity and method of using the same
SE0300791D0 (en) 2003-03-20 2003-03-20 Amersham Biosciences Ab Use of ph-responsive polymers
WO2004089997A1 (en) 2003-04-01 2004-10-21 Nitto Boseki Co., Ltd. Modified polyallylamine and process for producing the same
US6949625B2 (en) 2003-05-12 2005-09-27 Khorionyx Injectable implant of insoluble globin
WO2006085321A2 (en) 2005-02-10 2006-08-17 Affisink Biotechnology Ltd. Compositions and methods for purifying and crystallizing molecules of interest
IL157086A0 (en) 2003-07-24 2004-02-08 Guy Patchornik Multivalent ligand complexes
CA2560901C (en) 2003-08-29 2012-08-21 The University Of Newcastle Research Associates Limited Stimulant sensitive flocculation and consolidation
US7377686B2 (en) 2003-09-04 2008-05-27 Millipore Corporation Disposable mixing system
JP4231759B2 (en) 2003-09-22 2009-03-04 株式会社日立メディアエレクトロニクス Optical information recording device
DE10350248A1 (en) 2003-10-28 2005-06-16 Magnamedics Gmbh Thermosensitive, biocompatible polymer carriers with variable physical structure for therapy, diagnostics and analytics
US20090130704A1 (en) 2003-11-13 2009-05-21 Gyure Dale C Novel bioreactor
US7875448B2 (en) 2004-01-12 2011-01-25 Single Use Brx, Llc Bioreactor systems and disposable bioreactor
US20050282169A1 (en) 2004-01-29 2005-12-22 Turner Allen C Signatory sequences
US8105849B2 (en) 2004-02-27 2012-01-31 Board Of Regents, The University Of Texas System Integration of fluids and reagents into self-contained cartridges containing sensor elements
CA2559527C (en) 2004-03-12 2015-11-24 University Of Utah Cyclone reactor and associated methods
SE0400916D0 (en) 2004-04-05 2004-04-05 Amersham Biosciences Ab Polymeric ligands
EP1763575A4 (en) 2004-04-27 2009-09-23 Baxter Int Stirred-tank reactor system
JP4680990B2 (en) 2004-06-04 2011-05-11 エクセラーエックス, インコーポレイテッド Disposable bioreactor system and method
JP2008519277A (en) 2004-11-04 2008-06-05 バイオ−ラッド・パスツール Stackable chromatography module and chromatography column comprising a stack of such modules
US8288169B2 (en) 2005-01-21 2012-10-16 Argylla Technologies Surface mediated self-assembly of nanoparticles
JP4971997B2 (en) 2005-01-31 2012-07-11 メルク・シャープ・エンド・ドーム・コーポレイション Purification method of plasmid DNA
US8603805B2 (en) 2005-04-22 2013-12-10 Hyclone Laboratories, Inc. Gas spargers and related container systems
JP2006312117A (en) 2005-05-06 2006-11-16 Canon Inc Separation material for physiologically active substance and its production method
WO2006135673A2 (en) 2005-06-10 2006-12-21 Nanologix, Inc. Production of hydrogen gas and isolation of hydrogen producing microorganisms using replenishing coated substrates
WO2006138143A1 (en) 2005-06-15 2006-12-28 Amprotein Corporation Suspension culture vessels
JP4764264B2 (en) 2005-06-23 2011-08-31 本田技研工業株式会社 General engine electrical wiring structure
US20100267594A1 (en) 2005-06-24 2010-10-21 Rana Rohit K Nano-encapsulated triggered-release viscosity breakers
CN101296742A (en) 2005-08-26 2008-10-29 最佳空气控股公司 Method and device for separating a substance from a process gas
NL1030538C1 (en) 2005-11-28 2007-05-30 Eurocore Trading & Consultancy Device for indirectly cooling an air stream through evaporation.
CA2625499A1 (en) 2005-09-27 2007-04-05 Lance Liotta Method of isolating analytes from a sample
EP1951741A4 (en) 2005-10-27 2011-06-01 Life Technologies Corp Surface modification in a manipulation chamber
JP2009521672A (en) 2005-12-22 2009-06-04 ジーイー・ヘルスケア・バイオサイエンス・アクチボラグ Biomolecule preparation
EP1832341A1 (en) 2006-03-10 2007-09-12 MPG Max-Planck-Gesellschaft zur Förderung der Wissenschaften e.V. Highly efficient desalination and ion exchange using a thermoreversible polymer
WO2007109584A1 (en) 2006-03-16 2007-09-27 University Of Washington Temperature-and ph-responsive polymer compositions
AU2007262448A1 (en) 2006-04-14 2007-12-27 Interface Biologics Incorporated Grafted polymers and uses thereof
WO2007121981A1 (en) 2006-04-24 2007-11-01 Ciba Holding Inc. Cationic polysaccharide, its preparation and use
EP1873205A1 (en) 2006-06-12 2008-01-02 Corning Incorporated Thermo-responsive blends and uses thereof
FR2902799B1 (en) 2006-06-27 2012-10-26 Millipore Corp METHOD AND UNIT FOR PREPARING A SAMPLE FOR THE MICROBIOLOGICAL ANALYSIS OF A LIQUID
WO2008004988A1 (en) 2006-07-06 2008-01-10 Agency For Science, Technology And Research Thermally responsive micelles
US20080032396A1 (en) 2006-08-02 2008-02-07 Becton, Dickinson And Company Bioreactor and Method
US7935518B2 (en) 2006-09-27 2011-05-03 Alessandra Luchini Smart hydrogel particles for biomarker harvesting
EP1923461A1 (en) 2006-11-15 2008-05-21 Millipore Corporation A bioreactor
US8057092B2 (en) 2006-11-30 2011-11-15 Corning Incorporated Disposable spinner flask
US8569464B2 (en) 2006-12-21 2013-10-29 Emd Millipore Corporation Purification of proteins
US8362217B2 (en) 2006-12-21 2013-01-29 Emd Millipore Corporation Purification of proteins
ZA200904482B (en) 2007-01-22 2010-09-29 Genentech Inc Polyelectrolyte precipitation and purification of antibodies
US8137559B2 (en) 2007-02-09 2012-03-20 Ge Healthcare Bio-Sciences Ab Liquid clarification
EP2117587B1 (en) 2007-03-05 2018-01-10 OM Pharma Bacterial extract for digestive or urinary tract disorders and process for its preparation
US7981688B2 (en) 2007-03-08 2011-07-19 University Of Washington Stimuli-responsive magnetic nanoparticles and related methods
US20080284163A1 (en) 2007-05-15 2008-11-20 Millipore Corporation Connector for flexible tubing
US8105493B2 (en) 2007-06-29 2012-01-31 Jnc Corporation Aggregation and dispersion methods of magnetic particles, separation and detection methods using the same and detection kit
US9433922B2 (en) 2007-08-14 2016-09-06 Emd Millipore Corporation Media for membrane ion exchange chromatography based on polymeric primary amines, sorption device containing that media, and chromatography scheme and purification method using the same
US20110045081A1 (en) 2007-10-12 2011-02-24 Benedikt Steitz Magnetic, paramagnetic and/or superparamagnetic nanoparticles
WO2009089570A1 (en) 2008-01-14 2009-07-23 The University Of Melbourne Flotation aids and processes for using the same
EP2274361B1 (en) 2008-04-18 2016-10-12 S.P.C.M. Sa Functionalized cationic polyamines and their use to reduce the ndma formation during the treatment of aqueous systems, and applications in the water treatment industry, including wastewater and drinking water treatment processes
GB0809404D0 (en) 2008-05-23 2008-07-02 Univ Sheffield Method
US20110065900A1 (en) 2008-05-30 2011-03-17 Ge Healthcare Bio-Science Ab Separation method utilizing polyallylamine ligands
WO2009151514A1 (en) 2008-06-11 2009-12-17 Millipore Corporation Stirred tank bioreactor
WO2009158606A2 (en) 2008-06-27 2009-12-30 Stc.Unm Structure, synthesis, and applications for oligo phenylene ethynylenes
FR2934049B1 (en) 2008-07-16 2010-10-15 Millipore Corp UNIT AND METHOD FOR PREPARING A SAMPLE FOR THE MICROBIOLOGICAL ANALYSIS OF A LIQUID
AU2008221604B2 (en) 2008-09-22 2010-04-22 Commonwealth Scientific And Industrial Research Organisation Temperature-responsive polymer particles in protein separation applications
JP2012512244A (en) 2008-12-16 2012-05-31 イー・エム・デイー・ミリポア・コーポレイシヨン Protein purification
EP2370561B1 (en) 2008-12-16 2019-08-07 EMD Millipore Corporation Stirred tank reactor and method
CN102272146A (en) 2009-01-13 2011-12-07 通用电气健康护理生物科学股份公司 Precipitation of biomolecules with negatively charged polymers
PT2391660E (en) 2009-01-30 2014-11-03 Solenis Technologies Cayman Lp Quaternary vinylamine-containing polymers as additives in papermaking
US9080933B2 (en) 2009-11-09 2015-07-14 University Of Washington Through Its Center For Commercialization Stimuli-responsive polymer diagnostic assay comprising magnetic nanoparticles and capture conjugates
US20120077249A1 (en) 2010-04-20 2012-03-29 Millipore Corporation Separation Of Virus And/Or Protein From Nucleic Acids By Primary Amines
KR101827855B1 (en) * 2010-05-17 2018-02-12 이엠디 밀리포어 코포레이션 Stimulus responsive polymers for the purification of biomolecules
KR20130041843A (en) 2010-06-08 2013-04-25 이엠디 밀리포어 코포레이션 Methods of detecting residual amounts of polymers used in the purification of biomolecules
US20120283419A1 (en) 2011-05-03 2012-11-08 Avantor Performance Materials, Inc. Separation of protein monomers from aggregates by solid weak anion exchange support functionalized with amine moieties
US20140263011A1 (en) 2011-05-03 2014-09-18 Avantor Performance Materials, Inc. Novel chromatographic media based on allylamine and its derivative for protein purification
WO2014003137A1 (en) 2012-06-27 2014-01-03 旭化成メディカル株式会社 High-affinity antibody, and production method therefor
EP2682168A1 (en) 2012-07-02 2014-01-08 Millipore Corporation Purification of biological molecules
US20150218208A1 (en) 2012-08-27 2015-08-06 Asahi Kasei Medical Co., Ltd. Method for purifying antibody by temperature-responsive chromatography
AR093330A1 (en) 2012-11-01 2015-06-03 Novozymes As METHOD FOR DNA REMOVAL
US9938520B2 (en) 2012-12-11 2018-04-10 Qiagen Gmbh Preparation of silica particles
KR20150115745A (en) 2013-02-06 2015-10-14 에이전시 포 사이언스, 테크놀로지 앤드 리서치 Methods for reducing aggregate content in protein preparations
WO2014123485A1 (en) 2013-02-06 2014-08-14 Agency For Science, Technology And Research Protein purification methods
SG11201505203XA (en) 2013-02-26 2015-08-28 Agency Science Tech & Res Protein purification in the presence of nonionic organic polymers and electropositive surfaces
EP2961761B1 (en) 2013-02-28 2018-09-12 Agency For Science, Technology And Research Protein purification in the presence of nonionic organic polymers at elevated conductivity
US9994611B2 (en) 2013-02-28 2018-06-12 Agency For Science, Technology And Research Chromatographic purification of antibodies from chromatin-deficient cell culture harvests

Patent Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20040127639A1 (en) * 2001-03-30 2004-07-01 Xiaoru Wang Composite colorant particles
CN101420991A (en) * 2003-11-20 2009-04-29 血管技术国际股份公司 Polymer compositions and methods for their use
CN1938348A (en) * 2004-03-31 2007-03-28 巴斯福股份公司 Polymers comprising diallylamines
US20060281847A1 (en) * 2005-06-10 2006-12-14 Canon Finetech Inc. Fine particle liquid dispersion and recording medium using the same
CN101437901A (en) * 2006-03-03 2009-05-20 罗地亚管理公司 Modification of solid surfaces by means of polymer combinations
WO2008079302A2 (en) * 2006-12-21 2008-07-03 Millipore Corporation Purification of proteins
CN101678318A (en) * 2007-05-25 2010-03-24 默克专利股份公司 Graft copolymer for cation-exchange chromatography
CN111356434A (en) * 2017-12-12 2020-06-30 莱雅公司 Composition comprising an oil and a polyionic complex comprising a cellulose-based cationic polymer having at least one fatty chain

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
TOSHIHIRO SEO ET AL.,: "Self-Organization of Poly(allylamine)s Containing Hydrophobic Groups and Its Effect on the Interaction with Small Molecules. 1. Static Fluorometry", 《MACROMOLECULES》 *
赵文善等: "二甲基烯丙基对乙基苄基氯化铵的合成及表征", 《商丘师范学院学报》 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109574296A (en) * 2018-11-27 2019-04-05 合肥工业大学 A method of from recycling albumen in proteinic wastewater containing crab meat
CN109574296B (en) * 2018-11-27 2021-05-07 合肥工业大学 Method for recovering protein from crab meat protein-containing wastewater

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